Dual Affinity Polypeptides for Purification

ABSTRACT

The present invention relates to a process for purification of a target biomolecule, comprising the steps: (a) contacting (i) a target biomolecule, (ii) a dual affinity polypeptide, and (iii) a solid support comprising a catching ligand, wherein the ratio between the equilibrium dissociation constants of the dual affinity polypeptide, [K D,t /K D,s ], is at least 10° at standard conditions; and (b) recovering the target biomolecule by elution.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form.The computer readable form is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to a process for purification of a targetbiomolecule on a solid support comprising the steps: (a) contacting (i)a target biomolecule, (ii) a dual affinity polypeptide, and (iii) asolid support comprising a catching ligand.

BACKGROUND OF THE INVENTION

Recovery and purification of therapeutic proteins accounts forapproximately 50% of the manufacturing cost of biological drugs. Thegeneral industrial purification process often includes a number of unitoperation steps, like extraction, precipitation, as well as anion- andcation-exchange chromatography. Affinity chromatography is the preferreddownstream process step due to its high recovery, yield and specificity,but the current cost and limitations of affinity chromatography is verysubstantial and in many cases prohibitive for a more general use of thisunit operation. For a general description of conventional purificationprocedures including affinity chromatography see e.g. Jason and Rydén1998 (Jason, J-C and Rydén, L., Protein Purification: Principles,high-Resolution, Methods and Applications, 2nd edition, Wiley & sonsInc. New York, 1998).

Conventional affinity chromatography is in general characterized byhaving a capturing ligand immobilised to a solid phase matrix. Theligand reversibly binds a target molecule present in a fluid such asliquid culture medium or serum. Target molecules are recovered bydissociating the complex at eluting conditions. Commercially availableaffinity matrices are in a ready to use format including capturingligands covalently attached to the matrices. In conventional affinitychromatography the dissociation constant, K_(D), between the ligand andthe target protein is in the range of about 10⁻⁵-10⁻⁷M. Interactionswith dissociation constants exceeding 10⁻¹⁰-10⁻¹¹M are often impossibleto use, as the conditions required to dissociate the complex are thenthe same as those that will result in denaturation of the targetproteins.

The prior art include alternative variations of affinity chromatographypurification methods described in the literature (Wilchek, M. andGorecki, M. (1973), A New Approach for isolation of Biologically ActiveCompounds by Affinity Chromatography: Isolation of Trypsin).

FEBS Letters. 31, 1, 149-152, describes antibodies immobilized on aninsoluble material. The antibodies have affinity for a certain ligandattached to a complex of two or more proteins, and are independent ofthe chemical, physical and biological properties of the complex itself.The immobilized antibody matrix serves as means for concentrating thecomplex. The adsorbed complex can then be recovered from the column byelution. The authors use the trypsin enzyme reacted withdinitrophenylated soybean trypsin inhibitor (DNP-STI) to form thecomplex. The complex is adsorbed to anti DNP-column and eluted underconditions that dissociate the antigen-antibody binding. The affinitycolumn is then ready for the next purification cycle. The target trypsinis obtained by separation of the trypsin enzyme-dinitrophenylatedsoybean trypsin inhibitor complex into its components in a later step.

This procedure is different from the present invention in that theaffinity column is reusable and it is the binding between theimmobilized agent and the linker that is dissociated during elution andnot the bond between linker and target biomolecule.

Another concept described by Hammarbergh, B. et al., (Proc. Natl. Acad.Sci USA, 86, 4367-4371 (1989)), is a fusion protein affinity approachand its use to express recombinant human insulin-like growth factor II.The procedure relates to a recombinant target protein of interest (X)fused between two different affinity protein tails (A and B). Theprotein (X) has a protease-sensitive site. A cell lysate containing therecombinant tripartite fusion protein is first passed through anaffinity column containing a tail B-specific ligand. A mixture offull-length protein and proteolytic fragments containing the C-terminalfusion protein region can thus be obtained. In a second passage througha tail A-specific affinity column, the degraded proteins flow throughwhile full-length fusion protein is retained. After site-specificcleavage of the tails, the protein of interest (X) is obtained bypassing the cleavage mixture through a mixed affinity column for tails Aand B and collecting the flow-through. The authors describe a procedureto obtain the target protein by expressing the target protein as anintegrated part in between a dual affinity protein construct.

This is different from the present invention as the described affinityprocedure requires two different affinity columns and that theimmobilized ligand on the column and the dual affinity fusion protein isdissociated to recover the target biomolecule. Following the elutionstep and a regeneration procedure, the affinity columns are ready forthe next affinity purification cycle. The target protein is only part ofthe fusion protein and is obtained following enzymatic degrading steps.

In a review article by, Ford, C. F., Suominen, I, Glatz, C. E. (1991)Fusion Tails for the Recovery and Purification of Recombinant Proteins.Protein Expression and Purification, 2, 95-107, the authors discuss theapplications and advantages of using fusion tail systems to promoteefficient recovery and purification of recombinant proteins from crudecell extracts or culture media. In these systems, a target protein isgenetically engineered to contain a C- or N-terminal polypeptide tail,which provides the biochemical basis for specificity in recovery andpurification. Fusion tails are useful for enhancing recovery methods forindustrial downstream processing. Nevertheless, for the purification oftarget proteins a site for specific enzymatic cleavage is included,allowing removal of the tail after recovery. The article describes theapplication of fusion proteins with one binding partner having affinityfor the ligand immobilized on a matrix. The procedures include anenzymatic cleavage step to recover the target protein from the fusiontail as required.

This is different from the present invention as the described affinityprocedure requires that the fusion protein is dissociated from theligand immobilized on the column matrix to recover the protein.Following the elution step and a regeneration procedure, the affinitycolumn is ready for the next affinity purification cycle. Also,different from the present invention is that the target protein is partof the fusion protein and is only obtained following an enzymaticprocessing step.

In Rigaut, G. et al. (1991) (A Generic Protein Purification Method forProtein Complex Characterization and Proteom Exploration. NatureBiotechnology, 17, 1030-1032), is described a generic procedure forpurification of protein complexes using tandem affinity purification(TAP) tag. The purification requires one affinity step followed by anenzymatic step cleaving the first affinity tag from the complex and asecond affinity purification step to recover the target protein complexfrom the protease. Overall, the method involves two binding partners incombination both for binding to a ligand immobilized to a column matrixand a protease cleavage step to expose the second binding partner.

This is different from the present invention as the described affinityprocedure requires that the fusion protein is dissociated from theligand immobilized on the column matrix to recover protein. Followingthe elution step and a regeneration procedure, the affinity column isready for the next affinity purification cycle. Also, different from thepresent invention is that the target protein is part of the fusionprotein and is obtained following an enzymatic processing step.

EP1529844 describes a method for altering the properties of arecombinant target protein involving co-expression of target protein andthe binding partner. The target protein and the binding partner form acomplex in the cell. The complex formation result in altered propertiessuch as accumulation, stability and/or integrity, sub-cellularlocalization, post-translational modifications, purification, and phasepartitioning behavior of natural or recombinant target proteinsexpressed in a host organism. The binding partner may provide anaffinity tag that enables co-purification of the complex and the targetprotein contained therein. This description is different from thepresent invention as it describes a co-expression of the binder and thetarget in order to form a complex in the cell. The disclosed method isfor alteration of the target protein properties in general, whereas thepresent invention describes a dual affinity polypeptide specificallydesigned to facilitate a dedicated purification process, wherein thedual affinity polypeptides needs to possess specific binding properties.

Linder et al., (Linder, M., Nevanen, T., Söderholm, L., Bengs, O. andTeen, T., 1998, Biotechnology and Bioengineering, 60(5): 642-647)describes the use of CBD in fusionproteins for use as an affinity tagfor purification. Some leakage from the column was observed.

Shpigel, E. et al. (Biotechnol. Appl. Biochem. (2000) 31, 197-203,“Expression, purification and application of Staphylococcal Protein Afused to cellulose-binding domain”), describes an example of purifyingIgG using Protein A-CBD dual affinity polypeptide.

They claim that they save expensive coupling procedures by choosingimmobilization of the Protein A functionality to a solid phase throughthe cellulose-binding domain (CBD) of a fusion protein. The fusionprotein is immobilized on the column before adding the target.

Due to leakage problems this choice of dual affinity molecule isunsuitable for biopharmaceutical applications.

Sano et al. (U.S. Pat. No. 5,328,985) describes a fusion proteinconsisting of streptavidin and one or two immunoglobulin G (IgG) bindingdomains of protein A expressed in Escherichia coli. Thestrepavidin-protein A (ST-PA) fusion protein has functional biotin andIgG binding sites. Sano further describes complexes of thestreptavidin-protein A fusion protein, a monoclonal antibody to bovineserum albumin (BSA) and biotinylated horseradish peroxidise. Sano alsodescribes a method of labelling cell using the ST-PA fusion protein.Cells are incubated with an antibody to the cell surface antigen, Thy-1.The chimeric protein biotinylated marker complex is subsequently addedto the cell suspension. This technique was used to deliver biotinylatedFITC to the surface of the cells having Thy-1 antigens on their surface.

However, Sano does not describe or suggest using the ST-PA fusionprotein as a tool for purification purposes nor does he describe aprocedure of single use affinity chromatography column materials, norrecovery of a target protein.

WO 97/19957 describes an invention related to delivering toxins ornucleic acids into specific cell types using ST-PA fusion proteins forthe purpose. Similar to Sano et al. (vide supra), an antibody recognisea surface antigen on the cell surface. The ST-PA binds to the antibodyand facilitates a linkage to a biotinylated toxin bound to thebiotin-binding site. However, it is not described or suggested to usethe ST-PA fusion protein as a tool for purification purposes.

WO 01/95857 discloses a method and components for extracting toxicsubstances from mammalian blood. The method includes preparing anaffinity column (extracorporeal device) and a procedure forextracorporeal extraction of toxic material from mammalian body fluidsin connection with diagnosis or treatment of a mammalian condition ordisease. The extracorporeal affinity column exemplified in the patent ismade by coupling biotinylated entities to a matrix containingimmobilized avidin. The biotinylated entity includes a part that bindsstrongly to the toxin in the mammalian blood. The toxic material isremoved (i.e. immobilized but not recovered by elution from the column)from the blood following a conventional affinity chromatographyprocedure. The product from the flow through chromatography procedure ispurified blood as the target (toxic materials) stays immobilized on thecolumn after the process.

This is different from the present invention as it describes a procedurethat bind the target tightly with high affinity in order to removetarget from the product. The purification procedure is also differentfrom the present invention as the product does not bind to the affinitycolumn, but flows through and is collected as depleted from the toxicmaterial (the target). The toxic material is not released or recovered.

WO 97/09068 discloses a method and chemical components that alter theequilibrium dissociation constant between two pairs of bio-molecules.The chemical component is a polymer that can be stimulated to changeconformation and thus binding efficiency. The polymer is coupled e.g. toa specific site of the binding partner (the ligand) immobilised to thematrix of the affinity chromatography column. WO 97/09068 does notdescribe or suggest the use of a dual affinity component for affinitypurification, nor recovering of target molecules.

In general, methods that will improve the capturing efficiency andsimplify the purification process as well as reduce costs are desirable.

SUMMARY OF THE INVENTION

The present invention significantly improves and simplifies thedownstream processing and lowers the cost of affinity chromatographyprocesses in general. The present invention includes a generic capturingligand immobilised to a matrix, a target biomolecule and a semi genericdual affinity polypeptide with different binding affinity toward thetarget and the capturing ligand respectively. The dual affinitypolypeptide reacts with the target biomolecule to form a complex ofmedium binding affinity, and the complex binds non-covalently to ageneric affinity matrix with a strong binding affinity. The targetbiomolecule is recovered by specific elution from the generic matrixleaving the dual affinity polypeptide attached to the capturing ligandon the matrix, due to the tight binding to the ligand preventing leakagefrom the solid phase matrix.

In a first aspect the present invention provides a process forpurification of a target biomolecule, comprising the steps: (a)contacting (i) a target biomolecule, (ii) a dual affinity polypeptide,and (iii) a solid support comprising a catching ligand or dual affinitypolypeptide binding site, wherein the ratio between the equilibriumdissociation constants of the dual affinity polypeptide,[K_(D,t)/K_(D,s)], is at least 10⁰ at standard conditions; and (b)recovering the target biomolecule by elution.

In a second aspect the present invention provides a process forpurification of a target biomolecule, comprising the steps: (a)contacting (i) a target polypeptide, (ii) a dual affinity polypeptide,and (iii) a solid support comprising a catching ligand, wherein the dualaffinity polypeptide has an equilibrium dissociation constant, K_(D,t)towards the target biomolecule in the range from 10⁻² to 10⁻¹³ M, moreparticularly from 10⁻⁴ to 10⁻¹³ M at standard conditions, and whereinbinding of the dual affinity polypeptide to the catching ligand on thesolid support is provided by cleavage of a para-substituted benzylguanine resulting in a thioether bond; and (b) recovering the targetbiomolecule by elution.

DETAILED DESCRIPTION OF THE INVENTION

In conventional affinity chromatography the capturing ligand is attacheddirectly to the support. The main technical challenges are to optimizethe entire system with regard to e.g. ligand coupling, nature of thesupport material, flow, backpressure and physical dimensions of thecolumn. It should be understood that several of the technicallimitations in high performance affinity columns are closely linked,making performance and cost optimization as well as scale up difficult.The ligand, in traditional affinity chromatography, preferably possesthe following characteristics:

-   -   a) The ligand should have chemical properties that allow easy        covalent attachment to the matrix.    -   b) The ligand must be able to form a reversible complex with the        target molecule.    -   c) The specificity of the ligand's affinity for the target        molecule must be appropriate for the planned application.    -   d) The dissociation constant for the ligand-target molecule        complex under “loading conditions” should be strong enough to        enable formation of stable complexes or to give sufficient        retardation in the elution of the target molecule.    -   e) It should be easy to dissociate the ligand from the target        molecule by changing the conditions, e.g. pH or salt        concentration, without irreversibly damaging either.

Furthermore, in traditional affinity chromatography, the ligand isnormally covalently attached to the matrix and is also the componentbinding to the target molecule.

The capacity and quality of purification is greatly influenced by thecontact time between target and ligand in the affinity column, theso-called residence time.

In addition to the association rate of the target protein to animmobilized ligand, diffusion into the pores within the chromatographybeads in the column and mass transfer of the protein from the solutewill impact the dynamic binding capacity of a chromatography matrix.

The mass transfer of the target protein from the solute depends on avariety of factors, including type and degree of cross-linking,compressibility of the support material, the size of the pores and thephysical size of the target protein.

Flow rates, protein concentrations, column length, temperature, buffer,conductivity, and pH can also influence on pore diffusion and thedynamic binding capacity of the adsorbent.

Due to the requirement for rapid development of downstream processes andregulatory constraints, the residence time for a particular type ofbiological product such as for example a therapeutic antibody istypically fixed in the early development. Therefore, often the flow ratein the large scale column with e.g. larger bed height is tried adjustedto maintain the desired residence time used during the small scaledevelopment.

Due to technical constraints and the major investment required topurchase process-scale chromatography equipment, the scale up oftraditional high performance affinity chromatography is a majorchallenge.

The present invention suggests a more simple and flexible scale upprocess with less technical constraints.

Furthermore, conventional affinity chromatography is characterised byregeneration procedures to provide repeated uses of the columnmaterials. These cleaning procedures require extensive validation toallow multiple uses of the column.

The present invention differs in several aspects from the conventionalaffinity chromatography e.g.

-   -   the immobilized ligand binds tightly to the dual affinity        polypeptide (DAP) in order to prevent dissociation at elution        conditions    -   it is intended for single use applications

It is clear from the above that the role of the ligand in the presentinvention is to bind the DAP molecule and not the target molecule.

The attractive benefit of affinity chromatography is that it provides alarge increase in purity with a minimal loss of target molecule materialin a single unit operation. However, affinity chromatography is alsocharacterised by the high cost prohibiting the use of large columns andthus favouring repetitive use of smaller columns. This leads to extendedproduction processes and capacity loss proportional to the number ofcolumn reuses, increased loss and/or modification of the targetmolecule. In principle a typical affinity chromatography matrix can beused for up to 100 or more runs, but the average number of runs inmanufacturing scale appears to be several fold lower. One of the reasonsthat the matrix is discarded long before the end of its theoreticallifespan is that the affinity columns used in manufacturing aredimensioned to process the entire fermentation batch in far less than100 runs—in order to save cost, but also reduce the risk ofcontamination and handling failures. Rather than using the same matrixfor several fermentation batches, the matrix may be discarded afterprocessing of one fermentation batch, which leads to the relatively lownumber of average runs on an affinity matrix.

Controlling the flow rate through an affinity chromatography support isimportant in achieving binding. Flow rate through the column support isinextricably related to the efficiency of the separation; too fast aflow will cause the mobile phase to move past the beads faster than thediffusion time necessary to reach the internal bead volume. For eachapplication a flow rate can be selected to achieve an optimal balancebetween efficient binding and elution of the target protein and a fastseparation. Gravity driven flow chromatography is very slow andresolution of the protein separation can be adversely affected bysecondary diffusion effects. Therefore modern systems have activepumping to control flow rates and continuous monitoring of back pressureto ensure that the maximum operating back pressure is not beingexceeded.

In conventional columns fouling is of major concern. Debris, proteinsand salts can slowly build up fouling layers in the channels of highperformance affinity chromatography supports resulting in changed flowrates, reduced mass transfer rates, increased back pressure and hiddenand deactivated affinity ligands. Especially lipids and lipoproteinsmaterial can rapidly clog chromatography columns and it is oftennecessary to remove them before affinity purification. This isespecially important for samples derived from ascites fluid.

This pre purification step can be done by precipitation steps with forexample dextran sulphate and polyvinylpyrrolidine followed bycentrifugation and dialysis or desalting. The step can result in theloss of 5-10% of the target protein.

Omitting the delipidation step may be possible for the affinitypurification system of the present invention as the column is singleuse. This will result in a higher overall target recovery and a moreefficient downstream processing workflow.

Because of the intended single use of the columns according to theinvention the elution step is also simplified. As the support materialof the invention is not to be reused, one can more freely select elutionconditions. For example, it is possible to select any elution bufferswith an unconventional high concentration of salt, strong chaotropes,organic solvents etc. which will allow the recovery of the intact targetand leave the DAP molecule attached to the support. It is of noimportance if the properties of the support material are irreversiblychanged with respect to e.g. structure and flow characteristics andcannot be reused.

This flexibility in selecting elution conditions is often not possiblewhen using traditional high-performance affinity purification systems asthe internal structures and surfaces are highly optimized and sensitiveto polymer swelling or precipitation. Additionally, due to the cost oftraditional affinity columns, the operators can be reluctant to test newelution conditions further reducing the flexibility in elutionoptimization.

The traditional operation of affinity purification includes cycles ofequilibration, sample loading, elution and cleaning-in-place (CIP).

The cleaning steps or sanitization protocols have to be designed foreach specific target purification. As described above, a major concernduring operation is the build up of fouling layers or crosscontamination between runs.

The cleaning step often includes using chemically harsh buffers like 0.1M NaOH/1 M NaCl or 0.1 M phosphoric acid in a combination with sodiumchloride or ethanol, followed by regeneration. It is well establishedthat in general the dynamic binding capacity decreases as the number ofCIP cycles increases. Therefore, one needs to find an optimum betweenthe quality of the purified target, the number of runs, CIP's and thesize and cost of the particular column. Further, the change ofpurification quality needs to be monitored for most pharmaceuticalpurifications.

As suggested by the present invention, the cost of this qualityvalidation and the optimization of the CIP and runs can be greatlyreduced.

In the conventional affinity chromatography outlined above forpurification of e.g. monoclonal antibodies, the capturing ligand(Protein A) is attached to a solid phase matrix and has the affinitytowards the target biomolecule (monoclonal antibodies). The presentinvention provides advantages compared to conventional affinitypurification technologies for the downstream processing industry due tolower costs, high specificity and ease of use without compromising thequality of the down stream process. An essential feature of the presentinvention is the use of a dual affinity polypeptide as a linker betweenthe target molecule and the solid support comprising a ligand. Thesedual affinity polypeptides are particularly useful for the downstreamprocessing of biopharmaceutical and diagnostic proteins and peptides.

The invention suggests the improvement of the entire method of affinitypurification by eliminating several of the constraints in currentsystems.

By using the dual affinity polypeptide (DAP) and the generic supports ofthe invention, the majority of the above problems and limitations can becompletely eliminated or reduced.

According to the present invention the dual affinity purificationtechnology is characterized by a generic solid support, which in oneembodiment is a solid phase matrix, plus ready-to-use specific dualaffinity polypeptides serving as linker molecules. A dual affinitypolypeptide reacts with the target biomolecule. The dual affinitypolypeptide—target biomolecule complex subsequently connectsnon-covalently to a capturing ligand immobilized on a solid support bycontacting the complex and the solid support. The target biomolecule isrecovered by specific elution. The dual affinity polypeptide remainsattached to the ligand on the solid support during elution.

In one aspect the present invention therefore relates to a process forpurification of a target biomolecule, comprising the steps: (a)contacting (i) a target biomolecule, (ii) a dual affinity polypeptide,and (iii) a solid support comprising a catching ligand or dual affinitypolypeptide binding site, wherein the ratio between the equilibriumdissociation constants of the dual affinity polypeptide,[K_(D,t)/K_(D,s)], is at least 10° at standard conditions; and (b)recovering the target biomolecule by elution.

The dual affinity polypeptide acts as the linking partner between thesolid support and the target molecule. In one particular embodiment theaffinity of the dual affinity polypeptide towards the immobilized ligandis stronger than the affinity towards the target molecule. Furthermorethis difference in binding affinity, can be expressed as the ratiobetween the equilibrium dissociation constants. In one embodiment thisration is at least 1.

The dual affinity polypeptide according to the invention comprises atleast two binding sites, of which one binding site has affinity for theligand and another binding site has affinity for the target molecule.These binding sites are polypeptide based meaning that they compriseeither complete proteins or fragments of proteins. Such fragments shouldat least comprise the part of the protein containing the binding sitefor the specific target. The dual affinity polypeptide could be a fusionpolypeptide or could be two or more polypeptides chemically linked inany suitable way e.g. by a linker segment.

Therefore the present invention in further embodiments relates to a dualaffinity polypeptide having an equilibrium dissociation constant towardsa target biomolecule, K_(D,t) in the range from 10⁻² to 10⁻¹³ M, e.g.10⁻⁸ M, and an equilibrium dissociation constant towards a catchingligand, K_(D,s) in the range from 10⁻⁶ to 10⁻¹⁶ M, e.g. 10⁻¹⁰ M, and atthe same time the ratio, K_(D,t)/K_(D,s), should be matched such thatthe ratio is at least 10°, more particularly at least 10¹, moreparticularly 10², more particularly 10³ and even more particularly 10⁴

The above in other words means that binding of DAP to the target is inpreferred embodiments weaker than binding of DAP to the ligand.

Particularly the said dual affinity polypeptide has an equilibriumdissociation constant, K_(D,t) towards the target polypeptide in therange from 10⁻⁴ to 10⁻¹³ M, more particularly in the range from 10⁻⁶ to10⁻¹³ M, and an equilibrium dissociation constant, K_(D,s) towards thecatching ligand in the range from 10⁻⁶ to 10⁻¹⁶ M, more particularly inthe range from 10⁻¹¹ to 10⁻¹⁶ M.

In general the binding towards the ligand or the column cannot be toostrong. Therefore the value at the upper end of the range is notimportant in respect of K_(D,s).

In the context of the present invention the equilibrium dissociationconstant are measured according to the reaction:

${A + B}\underset{k_{d}}{\overset{k_{a}}{\rightleftarrows}}{AB}$

A and B represents the binding partners: the target biomolecule and thedual affinity polypeptide or the dual affinity polypeptide and thecatching ligand immobilized on the solid phase matrix.

The rate constants for the reaction above represent the rate at whichthe two molecules A and B associates and dissociates

${{{Dissociation}\mspace{14mu} {rate}}\mspace{14mu} - \frac{\lbrack{AB}\rbrack}{t}} = {k_{d}\lbrack{AB}\rbrack}$${{Association}\mspace{14mu} {rate}\text{:}\mspace{14mu} \frac{\lbrack{AB}\rbrack}{t}} = {{k_{a}\lbrack A\rbrack}\lbrack B\rbrack}$

When the rates are equal at equilibrium k_(a)[A][B]=k_(d) [AB], whichgives

$\frac{k_{d}}{k_{a}} = {\frac{\lbrack A\rbrack \lbrack B\rbrack}{\lbrack{AB}\rbrack} = K_{D}}$$\frac{k_{a}}{k_{d}} = {\frac{\lbrack{AB}\rbrack}{\lbrack A\rbrack \lbrack B\rbrack} = K_{A}}$

The candidate binding domains to be employed in the dual affinitypolypeptide should be evaluated according to the apparent equilibriumdissociation constants based on the total binding affinity of each ofthe dual affinities in a given DAP molecule irrespective of whether itcontains one or several binding domains for each specificity(target/capturing ligand). If e.g. A and B represent protein A (has fourto five binding domains) and avidin (having four binding sites)respectively the above ranges should apply for one protein A moleculefused to one avidin molecule. However, this does not exclude thepossibility that e.g. the DAP molecule could be composed of severalbinding candidates for the target and several candidates for the ligandon the matrix. The DAP could e.g. in another embodiment consist of 3protein A molecules linked to one or more avidin molecules. Thereforethe specified ranges as defined above should in the context of thepresent invention be evaluated based on the apparent binding constantsfor the binding domains in common.

In the context of the present invention the specified equilibriumdissociation constants are determined by surface plasmon resonance (SPR)technology using a Biacore Instrument as illustrated in detail in theexamples. The conditions described herein represent the standardconditions. As a suitable starting point for selecting different bindingdomains to be combined in the DAP molecule published K_(D)'s may beused.

The two binding pairs should be selected based on the K_(D's) duringspecific binding conditions, but also considering the planned elutionconditions, when the target is recovered and the DAP molecule remains onthe support.

As described above determination of dissociation affinities of variousbinding domains in the context of a DAP molecule was accomplished byusing surface plasmon resonance (SPR). Such evaluation can be done withthe Biacore system. Biacore has commercial instrumentation wheremeasurements based on SPR make determinations on protein-proteininteractions. The evaluation was conducted having the complete DAPimmobilized on the sensor chip used in the Biacore instrument. TheBiacore system defines the characteristics of proteins in terms of theirspecificity of interaction with other molecules, the rates at which theyinteract (association and dissociation), and their affinity (how tightlythey bind to another molecule). This technique has been described e.g.for determining the binding intereactions between specific antibodiesand their target (see e.g. Rönnmark, 2002, Eur. J. Biochem., 269:2647-2655).

In the examples below several DAP candidates have been evaluated andtheir binding affinities under standard conditions (as described in theexamples) have been measured for the complete DAP. Other methods mayalso be used, however, results may then differ. A list of alternativemethods has been described below.

Quantitative measurement of non-covalent protein-ligand interactions iswell known. The methods suited for quantitative measurement of bindingconstants of particular relevance for the present invention includevarious versions of surface plasmon resonance (SPR) and circulardichroism (CD).

Other methods include mass spectrometry methods for dynamic titrationslike ESI-MS titration, HPLC-ESI-MS titration or MALDI-SUPREX titration.

Other methods are based on determining the dissociation constant of aligand at a binding site indirectly by competitive displacement of aradioactive ligand or by measurement of NMR chemical shift as functionof concentration, fluorescence spectroscopy analysis of e.g. signalquenching, X-ray crystallographic measurement of the ligand occupancy,isothermal calorimetry (ITC) or enzyme inhibition.

Yet other methods use labeled ligands, for example capillaryelectrophoresis with laser-induced fluorescence detection of enzymelabeled ligands.

Alternatively, binding constants can be found from computationaltechniques by using de novo design, data mining and sophisticatedalgorithms.

In the context of the present invention the appropriate ranges for theequilibrium dissociation constants as specified in the claims shouldapply to the complete dual affinity polypeptide and not to theindividual binding parts measured separately.

Moreover, if a single candidate binding domain has a weaker bindingaffinity towards the target or ligand than required according to thepresent invention, it still could be applicable by combining severalsuch candidate binding domains into one DAP.

This is due to the valence effect. It is possible to obtain an increasedbinding strength due to an avidity gain. Single domains with a lowintrinsic affinity combined into multimers often generates avidityeffects which lead to slower dissociation rates and increased functionalaffinities by more than 100 fold (MacKenzie, C. R. et al (1996),Analysis by surface plasmon resonance of the influence of valence on theligand binding affinities and kinetics of and anti carbohydrateantibody. Journal of Biological Chemistry, 271, 1527-1533). It ispossible to measure effects from monovalent and bivalent bindings, butat higher binding valences the situation becomes so complex that it isimpossible to distinguish between different binding valances.Nevertheless relative data can be obtained and are used in the contextof the present invention

The invention provides a purification procedure wherein the firstreaction between the target molecule and the dual affinity polypeptidein one particular embodiment can be completed in free solution. Reactionbinding kinetics is about 1000 times faster in free solution compared tointerface reactions (Nygren, H. and Stenberg, M. (1989) Immunochemistryat interfaces. Immunology, 66, 321-327).

The target molecule-dual affinity polypeptide complexes are subsequentlypresented to and bind efficiently to the ligand on the solid support.The strong binding (fast association rate and slow dissociation rate ofthe ligand towards the dual affinity polypeptide) depletes the mobilephase of target-DAP complexes. The target molecules are recovered fromsolution through this sequential procedure facilitated by the secondbinding functionality of the dual affinity polypeptide.

Due to the described differences in equilibrium dissociation constantsthe target polypeptide can be efficiently eluted without eluting thedual affinity polypeptide. Elution can in one embodiment be performed bychanging either pH, ionic strength or chaotropic ions in solution, orany combination thereof.

The K_(D) value can be influenced by changing conditions like pH, ionicstrength, temperature and polar properties. Unfortunately, theliterature values for K_(D) are not always listed at relevant elutionconditions. Though, the skilled in the art will be able to find elutionconditions which will only break the weakest binding without disturbingthe stronger binding in cases were the binding to the solid matrix issufficiently strong (i.e. K_(D,s)<10⁻⁹ M and the ratio between K_(D)values is at least 1 when measured at standard binding conditions).

The criteria for selecting the target specific binding pairs of theinvention resemble those for the traditional affinity chromatographywith regard to dissociation constant, specificity, binding and possibleelution conditions. However, since elution conditions are usuallydifferent from the conditions applied when measuring K_(D)'s on theBiacore instrument in the present invention the limits set for theapplicable ranges of the two distinct binding affinities of the DAP hasbeen determined under standard conditions, which equals the conditionsused in the examples.

The criteria for selecting the specific ligand binding domains of theinvention are somewhat different from the criteria used in thetraditional affinity chromotography, as the DAP molecule is not to beeluted from the support.

Binding domains which are specific and strong, but cannot be brokenunder normal elution conditions are not suited for traditional affinitychromatography. Such binding domains can be used in the presentinvention. Examples include the very specific biotin-Streptavidinbinding, which for most practical applications cannot be reversed underelution conditions and consequently is well suited as one of the bindingpairs of the invention.

In general, the binding between DAP and the ligand should be strongerthan the binding between DAP and the target and strong enough to preventleakage of the DAP molecule from the support during elution of thetarget.

Preferred ligand-DAP binding pairs are strong and exhibit no or littlereduction in binding strength due to changing pH, ionic strength,solvents, chaotropic agents, temperature etc.

It should be clear that when changing the scale of purification, usingthe system of the invention, the amount of DAP added is adjusted to theamount and concentration of target protein. As the DAP molecule can besupplied as a concentrate, the binding conditions can be adjusted withrespect to e.g. pH and salts. Also, the temperature and time can beselected to give the best binding and subsequently purification.

The size and capacity of the generic column is selected to be largeenough to capture the DAP molecules. Potentially several columns areused in parallel or in a bundle.

If purifying another target, another appropriate DAP molecule isselected. The same or another column can be used.

In one embodiment the dual affinity polypeptide is a fusion polypeptide.Such fusion polypeptides can either be prepared by chemically linkingtwo appropriate proteins or alternatively in another embodiment thefusion protein can be synthesized as a recombinant polypeptide. Thefusion polypeptide can be linked in any suitable way e.g. by a linkersegment. and the fusion polypeptide should at least comprise the bindingdomains of the selected proteins. The linker peptide should be selectedin such a way that it is not unstable resulting in degradation. Thelinker could e.g. be a highly O-glycosylated linker as linkers betweencatalytic domains and carbohydrate binding domains known from fungalcarbohydrases, or it could be proline rich linkers.

The dual affinity polypeptide comprises at least one binding domaincapable of binding to the target biomolecule with the desired bindingspecificity as described. The binding domain can be comprised in thecomplete protein or it can be a fragment of the protein which hasretained its binding specificity. Many proteins have been described inthe literature displaying affinity towards biomolecules, e.g. peptides,proteins, DNA, RNA, carbohydrates, and all such proteins or fragmentsthereof are potentially useful in the context of the present inventionas candidates for the dual affinity polypeptide.

The said binding domain directed towards the target biomolecule can inone embodiment therefore be selected from but not limited to the groupconsisting of protein A, protein A fragments, protein A derived domains(e.g. domains known as an Affibody®), antibodies, antibody fragments,lipocalins, and lectins.

Combinatorial protein engineering has been applied to develop artificialproteins that can bind to selected targets with high affinity and beused as alternatives to antibodies (Nygren, P.-Å. & Skerra, A. (2004).Binding proteins from alternative scaffolds. J. Immunol. Methods, 290,p. 3-28; Binz, H. K. & Pluckthun, A. (2005). Engineered proteins asspecific binding reagents. Curr. Opin. Biotech. 16, p. 459-469). In thecontext of the present invention the term “affibody” defines a class ofengineered proteins selected for their specific binding activity towardsa desired target and based on the Z domain, which is a 58 residuethree-helical bundle derived by a single amino acid substitution in theB domain of staphylococcal protein A (SPA) (Nilsson, B., Moks, T.,Jansson, B., Abrahmsén, L., Elmblad, A., Holmgren, E. et al. (1987)Protein Eng. 1, p. 107-113). The Z domain binds to the Fc region ofimmunoglobulins as do the five homologous SPA domains, but unlike theparental domain it does not bind to the Fab region. Such affibodies areexamples of a protein A derived binding domain.

The dual affinity polypeptide also comprises at least one binding domaincapable of binding to the catching ligand immobilized on the solidsupport. This second binding domain can be comprised in the completeprotein or it can be a fragment of the protein which has retained itsbinding specificity. In one embodiment the second binding domain isselected from but not limited to the group consisting of avidin,streptavidin, neutravidin, steroid receptor, antibody, antibodyfragment, amyloglucosidase (AMG), enzyme domain (e.g. cellulose bindingdomain, CBD), lipocalins, and lectins. As stated above these candidates,for the second binding domain, are meant as examples illustrating theinvention, however, these examples should not be seen as the only usablecombinations.

In one embodiment the antibody is selected from the group consisting ofLlama and camel antibodies.

In a particular embodiment the dual affinity polypeptide according tothe invention comprises at least one binding domain of protein A fusedto at least one biotin binding domain of avidin, streptavidin orneutravidin.

In a particular embodiment the dual affinity polypeptide according tothe invention comprises at least one binding domain of a protein Aderived binding domain fused to at least one biotin binding domain ofavidin, streptavidin or neutravidin.

In another particular embodiment the dual affinity polypeptide comprisesat least one binding domain of an affibody fused to at least one biotinbinding domain of avidin, streptavidin or neutravidin.

In another particular embodiment the dual affinity polypeptide comprisesat least one binding domain of an antibody fused to at least one biotinbinding domain of avidin, streptavidin or neutravidin.

In another particular embodiment the dual affinity polypeptide comprisesat least one binding domain of protein A fused to AMG, CBD or(VhhRR6(R2)).

In another particular embodiment the dual affinity polypeptide comprisesat least one binding domain of a protein A derived binding domain fusedto AMG, CBD or (VhhRR6(R2)).

In another particular embodiment the dual affinity polypeptide comprisesat least one binding domain of an affibody fused to AMG, CBD or(VhhRR6(R2)).

In another particular embodiment the dual affinity polypeptide comprisesat least one binding domain of an antibody fused to AMG, CBD or(VhhRR6(R2)).

The dual affinity polypeptide can as illustrated in the examples belinked chemically; however, a more cost efficient way to produce thedual affinity polypeptide would be to express it as a recombinant fusionprotein.

In one embodiment of the invention, the fusion polypeptide is producedas a recombinant polypeptide.

Another possibility also envisioned would be to co-express the fusionprotein and the target biomolecule in the host cell making it possibleto load the crude cell culture extract directly on the solid support.

In a further embodiment the target biomolecule and the DAP is expressedseparately but in the same type of host cell.

In a particular embodiment the fusion protein is expressed as arecombinant protein, particularly the fusion protein is in oneembodiment recombinant Streptavidin linked to protein A. Such fusionprotein can be produced intracellular in E. coli as described in Sano(T. Sano and C. R. Cantor (1991) BioTechnology 9 p 1378-1381),preferentially using the construct pTSAPA-2 carrying two IgG bindingdomains. However this construct is not industrially feasible asintracellular production with recovery of inclusion bodies in E. coli donot give industrially relevant yields and the production process ishighly complex. A process based on a secreted fusion produced in e.g.Bacillus or Aspergillus is of much higher industrial relevance.

The nucleotide sequence encoding the fusion protein according to theinvention may preferably be expressed by inserting the nucleotidesequence or a nucleic acid construct comprising the sequence into anappropriate vector for expression. In creating the expression vector,the coding sequence is located in the vector so that the coding sequenceis operably linked with the appropriate control sequences forexpression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorwhich exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one which, when introduced into thehost cell, is integrated into the genome and replicated together withthe chromosome(s) into which it has been integrated. Furthermore, asingle vector or plasmid or two or more vectors or plasmids whichtogether contain the total DNA to be introduced into the genome of thehost cell, or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed cells. Aselectable marker is a gene the product of which provides for biocide orviral resistance, resistance to heavy metals, prototrophy to auxotrophs,and the like.

A conditionally essential gene may function as a non-antibioticselectable marker. Non-limiting examples of bacterial conditionallyessential non-antibiotic selectable markers are the dal genes fromBacillus subtilis, Bacillus licheniformis, or other Bacilli, that areonly essential when the bacterium is cultivated in the absence ofD-alanine. Also the genes encoding enzymes involved in the turnover ofUDP-galactose can function as conditionally essential markers in a cellwhen the cell is grown in the presence of galactose or grown in a mediumwhich gives rise to the presence of galactose. Non-limiting examples ofsuch genes are those from B. subtilis or B. licheniformis encodingUTP-dependent phosphorylase (EC 2.7.7.10), UDP-glucose-dependenturidylyltransferase (EC 2.7.7.12), or UDP-galactose epimerase (EC5.1.3.2). Also a xylose isomerase gene such as xyIA, of Bacilli can beused as selectable markers in cells grown in minimal medium with xyloseas sole carbon source. The genes necessary for utilizing gluconate,gntK, and gntP can also be used as selectable markers in cells grown inminimal medium with gluconate as sole carbon source. Other examples ofconditionally essential genes are known in the art. Antibioticselectable markers confer antibiotic resistance to such antibiotics asampicillin, kanamycin, chloramphenicol, erythromycin, tetracycline,neomycin, hygromycin or methotrexate.

Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3,TRP1, and URA3. Selectable markers for use in a filamentous fungal hostcell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell are theamdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae andthe bar gene of Streptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANS1 (Gems et al., 1991, Gene 98:61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention, which are advantageously usedin the recombinant production of the polypeptides. A vector comprising apolynucleotide of the present invention is introduced into a host cellso that the vector is maintained as a chromosomal integrant or as aself-replicating extra-chromosomal vector as described earlier. The term“host cell” encompasses any progeny of a parent cell that is notidentical to the parent cell due to mutations that occur duringreplication. The choice of a host cell will to a large extent dependupon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as grampositive bacteria including, but not limited to, a Bacillus cell, e.g.,Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacilluslautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium,Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis; or a Streptomyces cell, e.g., Streptomyces lividans andStreptomyces murinus, or gram negative bacteria such as E. coli andPseudomonas sp.

In a preferred aspect, the bacterial host cell is a Bacillus lentus,Bacillus licheniformis, Bacillus stearothermophilus, or Bacillussubtilis cell. In another preferred aspect, the Bacillus cell is analkalophilic Bacillus.

The introduction of a vector into a bacterial host cell may, forinstance, be effected by protoplast transformation (see, e.g., Chang andCohen, 1979, Molecular General Genetics 168: 111-115), using competentcells (see, e.g., Young and Spizizin, 1961, Journal of Bacteriology 81:823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of MolecularBiology 56: 209-221), electroporation (see, e.g., Shigekawa and Dower,1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5771-5278).

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensisor Saccharomyces oviformis cell. In another most preferred aspect, theyeast host cell is a Kluyveromyces lactis cell. In another mostpreferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filobasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,or Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, trans-formation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

The contact between the target biomolecule, the dual affinitypolypeptide (DAP), and the solid support can be performed in severaloptional ways. In one embodiment all components could be brought intocontact in one step, eg. by loading the target polypeptide and thefusion protein on the solid support without pre-incubation in solution.The dual affinity polypeptide can however, be contacted with the targetbefore loading this complex on the solid support. In this embodiment thetarget biomolecule and the dual affinity polypeptide are contactedfirst, e.g. in solution, and subsequently the formed complex iscontacted with the solid support. Depending on the nature of the solidsupport preferred embodiments of this principle could differ.

In one preferred embodiment the solid support is a solid phase matrix.This includes conventional solid phase matrixes. In the case of solidphase matrixes in the form of columns, the target and the dual affinitypolypeptide can in one embodiment be contacted first in solution andsubsequently contacted with the solid phase matrix by loading thecomplex onto the column.

It can be envisioned however, that e.g. the dual affinity polypeptidecan be loaded on the solid support first and subsequently loading thetarget biomolecule

In another embodiment the solid support is in the form of particles, inwhich case the order of contact is of less importance, and the contactof all the components could conveniently be performed in solution in onestep or in several steps.

The catching ligand according to the invention is covalently attached tothe solid support. As explained above the ligand according to thepresent invention is different from the ligand used in traditionalaffinity chromatography where the purpose of the ligand is to bind thetarget. In the present invention the ligand should bind to the DAP.Ligands are well known in the art and below are given examples that canbe applied according to the invention. In the context of the presentinvention in one particular embodiment instead of a ligand attached tothe solid phase the solid phase could alternatively comprise a bindingaffinity or binding site towards the DAP. An example could be celluloseas the solid phase and CBD (cellulose binding domain) as part of theDAP.

In one embodiment the ligand is chosen from but not limited to the groupconsisting of biotin, acarbose, steroids, haptens, epitope-peptides,dyes and enzyme inhibitors. In a particular embodiment the ligand isbiotin. The ligand can be chemically attached to the solid support asdescribed in the examples where the chemical attachment of acarbose andreactive red 6 is illustrated.

The coupling of affinity ligands to supports strongly influences thespecificity, capacity and cost of traditional affinity chromatographycolumns.

The current state of the art in covalent coupling technology allows forchemo and regio selective coupling of the binding ligands to thesupport, often using spacers or linkers to anchor the ligand to thesurface.

Great care is taken to avoid using functional groups that are close to abinding site or that play a role in the interaction between the ligandand target molecule.

If a suitable functional group does not exist on the ligand, furtherderivatizing of the ligand can be done to add an appropriate functionalgroup. Numerous references describe appropriate chemistries, including“Bioconjugate Techniques”, by Greg T. Hermanson, Academic Press, 2008and “Chemistry of Protein Conjugation and Cross-linking”, by Shan S.Wong, CRC Press, 1991.

It is commonly accepted that a high concentration of coupled ligandoften has adverse effects on affinity chromatography, also the bindingefficiency of the medium may be reduced due to steric hindrance betweenthe active sites. This is particularly pronounced when large moleculessuch as antibodies, antigens and enzymes interact with small ligands.

In addition, the target substances may become more strongly bound toclosely packed ligands making elution difficult and also the extent ofnonspecific binding increases at very high ligand concentrations, thusreducing the selectivity of the affinity column.

Ligand-surface interface interaction is known to be important for theaffinity ligand performance. The length of spacer arms between theligand and the surface is critical. If it is too short, the arm isineffective and the ligand fails to bind the target in the sample. If itis too long, proteins may bind non-specifically to the spacer arm andreduce the selectivity of the separation. Often 4-12 atom longhydrophilic arms are used.

MabSelect™ Media and HiTrap MabSelect™ (GE Healthcare) are examples ofaffinity columns using oriented coupling of recombinant Protein A to thematrix via an engineered C-terminal cysteine and a hydrophilic spacerarm.

The present invention suggests the use of soluble dual affinitypolypeptide which can be characterized and used in any concentrationappropriate for the specific target concentration. The technicalchallenge of coupling delicate target specific binding ligands to asolid support is substituted with more simple preparation of solublemolecules making it possible to utilize the entire arsenal of analyticalmethods.

There are numerous types of support material for affinitychromotography.

The size and uniformity of beads, the distribution of internal channelsand the nature of the surfaces has all been optimized to producenumerous types of supports.

In general, smaller particle size and greater porosity, ensuresincreased dynamic binding capacity. On the other hand, resistance tomechanical collapses is reduced.

Both compressible and the incompressible support material needs to berobust enough to survive multiple cycles without change of flow rateswhich will influence the residence time.

The solid support are in the form of beads, gels or granulates. Thequality of packing of the solid support material in columns fortraditional affinity purification and the flow rates during operationgreatly influence the performance.

Specialized equipment is used to successfully pack large columns above5-10 cm in diameter. High performance columns are normally purchasedpre-packed and in standard sizes. Consequently, the practical dimensionsduring scale up depend on available column systems for the purificationof the particular target molecule.

The present invention suggests a general method using target DAPmolecules and a generic column.

One of the most important factors in determining the cost and quality ofthe large scale purification is the chemical and mechanical stability ofthe adsorbent.

Traditional affinity columns with immobilized protein ligands aresusceptible to further degradation due to for example oxidation ormicrobial growth.

Therefore, due to the cost of large affinity columns, great care has tobe taken to control the storage condition between uses. Often the columnis washed and stored with a special buffer solution containing antimicrobial agents, alcohols or similar. These storage solutions must bewashed away before use.

Some affinity ligands are also sensitive to proteases and the columnlifetime will be reduced unless special cleaning and regenerationprocedures are followed rigorously. The freedom to design efficientaffinity purification procedures is therefore somewhat restricted.

A single use column system according to the invention or a column systemusing synthetic ligands will not have the above technical limitations.

In one embodiment of the invention the solid support is in the form of asolid phase matrix. The solid phase matrix may comprise, as the matrixbackbone, any natural or synthetic and organic or inorganic materialknown per se to be applicable in solid phase separation of proteins andother biomolecules, e.g. natural or synthetic polysaccharides such asagar-agar and agaroses; celluloses, cellulose ethers such ashydroxypropyl cellulose, carboxymethyl celluose; starches; gums such asguar gum, and gum arabic, gum ghatti, gum tragacanth, locust bean gum,xanthan gum; pectins; mucins; dextrans; chitins; chitosans; alginates;carrageenans; heparins; gelatins; synthetic polymers such as polyamidessuch as polyacrylamides and polymethacrylamides; polyimides; polyesters;polyethers; polymeric vinyl compounds such as polyvinylalcohols andpolystyrenes; polyalkenes; inorganic materials such as siliciousmaterials such as silicon dioxide including amorphous silica and quartz;silicas; metal silicates, controlled pore glasses and ceramics; metaloxides and sulfides, or combinations of these natural or synthetic andorganic or inorganic materials.

The matrix backbone is preferably selected from agar-agar, agaroses,celluloses, cellulose ethers such as hydroxypropyl cellulose,carboxymethyl cellulose, polyamides such as poly(meth)acrylamides,polyvinylalcohols, silicas, and controlled pore glasses.

Especially interesting solid phase materials as matrix backbones aree.g. agar or agarose beads such as Sepharose and Superose beads from GEHealthcare, USA, and Biogel A from Biorad, USA; dextran based beads suchas Sephadex, GE Healthcare; cellulose based beads and membranes such asPerloza cellulose from Iontosorb, Czech Republic; composite beads suchas Sephacryl and Superdex, GE Healthcare, USA; beads of syntheticorganic polymers such as Fractogel from Tosoh Lifesciences LLC, USA;POROS media from Applied Biosystems, USA, Bio-Rex, Bio-Gel P and MacroPrep from Biorad, HEMA and Separon from TESSEK and Hyper D and Trisacrylmedia from Pall Corporation, USA, Enzacryl and Azlactone, 3M, USA; beadsof siliceous materials such as controlled pore glass, PROSEP, fromMillipore, USA, and Spherocil, Pall Corporation, USA; and coated silicacomposites in the form of beads or membranes such as ACTI-DISK, ACTI-MODand CycloSep from Arbor Technologies, USA.

The ligand (e.g. biotin or similar specific molecules of low molecularweight (LMW)) is then covalently attached to this material. Severalcoupling chemistries of ligand molecules to the solid support can beselected from text books on the subject (Protein Purifuication, 1998,2ed, eds. Janson, J-C., Rydén, L, Wiley & sons inc. New York). Based onthe particular purification task the best candidate of ligandderivatives is coupled to the best choice of solid support, e.g. solidphase matrix or particles. Production process properties of the affinitysolid matrix are analyzed through practical laboratory and pilottesting.

The ligands may be attached to the solid phase material by any type ofcovalent bond known per se to be applicable for this purpose, either bya direct chemical reaction between the ligand and the solid phasematerial or by a preceding activation of the solid phase material or ofthe ligand with a suitable reagent known per se making it possible tolink the matrix backbone and the ligand. Examples of such suitableactivating reagents are epichlorohydrin, epibromohydrin, allylglycidylether; bis-epoxides such as butanedioldiglycidylether;halogen-substituted aliphatic compounds such as di-chloro-propanol,divinyl sulfone; carbonyldiimidazole; aldehydes such as glutaricdialdehyde; quinones; cyanogen bromide; periodates such assodium-meta-periodate; carbodiimides; chloro-triazines such as cyanuricchloride; sulfonyl chlorides such as tosyl chlorides and tresylchlorides; N-hydroxy succinimides; 2-fluoro-1-methylpyridiniumtoluene-4-sulfonates; oxazolones; maleimides; pyridyl disulfides; andhydrazides. Among these, the activating reagents leaving a spacer groupSP1 different from a single bond, e.g. epichlorohydrin, epibromohydrin,allylglycidylether; bis-epoxides; halogen-substituted aliphaticcompounds; divinyl sulfone; aldehydes; quinones; cyanogen bromide;chloro-triazines; oxazolones; maleimides; pyridyl disulfides; andhydrazides, are preferred.

In one embodiment the solid support is in the form of particles.Particles can be selected from the group comprising microspheres, latexparticles or beads. The particles can be made from but not limited tothe group consisting of e.g. polystyrene, silica, naphtaleen,polybutylmethacrylate.

The generic solid support can be produced at costs comparable to ionexchange matrices and the recombinant dual affinity protein can also beproduced as a recombinant fusion protein by fermentation at low cost.Due to the lowered cost of the novel downstream procedure materials, thepurification technology may be provided as disposables, which eliminatethe need for expensive cleaning in place (CIP) and certain validations.Another consequence of the reduced cost is optional large column-volumeapplications, which saves manufacturing labour expenses, preventrepeated purification re-runs and limit time occupations of thedownstream process plant.

The use of a generic solid support including the capturing ligand andthe potentially improved binding efficiency and capacity due to complexformation in solution poses several advantages over the conventionalaffinity chromatography. These advantages are listed below.

-   -   No time consuming and expensive chemical conjugation reactions,        purifications and QC procedures of protein ligands to prepare        the affinity column material prior to affinity purification of        the target molecule.    -   The generic matrix is more cost efficient to manufacture        compared to the present commercial affinity matrices matrices        (e.g. Protein A). Only one type of capturing column material is        required for all affinity purifications using the dual affinity        polypeptide principle. The low molecular weight ligand, e.g.        biotin, dye molecules or similar specific low molecular weight        (LMW) molecules are covalently attached by simple low cost        conjugation procedures to make the generic solid phase matrix.    -   The preferred fermentation of a dual affinity polypeptide fusion        protein is “simple”, based on known technology and provide the        conjugation needed between binding domains in DAP    -   The manufacturing cost of DAP molecules is comparable or cheaper        than recombinant Protein A molecules.    -   The DAP fusion protein and the generic matrix required for        purification purposes costs a fraction of the ready-to-use        Protein A affinity matrix for similar purposes    -   The DAP transport and immobilize the target molecules to the        generic matrix during the purification process. The is no need        for an expensive and time consuming immobilization of a        dedicated ligand to make an specific purification matrix as        known in conventional affinity chromatography.    -   The low cost of the components in the presented invention        facilitate a disposable affinity purification process featuring        -   elimination of CIP procedures        -   elimination of validation procedures        -   save time on regulatory issues        -   exclude repetitions of down stream process cycles        -   limit operational failures        -   lower labor expenses during processing        -   shorter manufacturing run time        -   limit risk of contamination        -   easy to use        -   lower capacity cost investments due to flexible plant            designs        -   better down stream process economy    -   Substitution of conventional multi-cycle protein separation        procedures to a single step using the disposable affinity        purification technology.

In a particular modified form of the invention it could be envisionedthat the DAP molecule could bind covalently to the solid support. Thiswould still allow the possibility of having the DAP and the targetreacting in solution. Such a covalent bond could in one embodiment beformed by cleavage of a para-substituted benzyl guanine resulting in athioether bond.

One embodiment of this modified form of the invention therefore relatesto a process for purification of a target biomolecule, comprising thesteps: (a) contacting (i) a target polypeptide, (ii) a dual affinitypolypeptide, and (iii) a solid support comprising a catching ligand,wherein the dual affinity polypeptide has an equilibrium dissociationconstant, K_(D,t) towards the target biomolecule in the range from 10⁻²to 10⁻¹³ M, more particularly from 10⁻⁴ to 10⁻¹³ M at standardconditions, and wherein binding of the dual affinity polypeptide to thecatching ligand on the solid support is provided by cleavage of apara-substituted benzyl guanine resulting in a thioether bond; and (b)recovering the target biomolecule by elution.

The basic principle of the affinity purification technology, forpurifying a target molecule (polyclonal antibody) is illustrated below.

EXAMPLES Example 1 Preparation of Dual Affinity Linker by ChemicalConjugation

Based on published values for binding affinities, dual linker bindingfunctionalities were selected that fit both to the binding to the ligandmatrix (K_(D,s)˜10⁻⁹ to 10⁻¹⁶M) and to the target biomolecules(products, K_(D,t)˜10⁻² to 10⁻¹³ M). To investigate the influence of theK_(D,s), some components with K_(D,s)-values outside the above intervalwere also tested.

In order to prepare a conjugate made from Protein A and a biotin bindingprotein e.g. Avidin, Streptavidin or Neutravidin the two proteins werechemically activated separately as a first step and joined together bycross linking in a second step afterwards.

Protein A do not have accessible sulphydryl (—SH) on the surface, sothese were introduced be reaction with SATA (N-succinimidylS-acetylthioacetate) to primary amine (—NH2) functional groups onProtein A. SATA (or SATP) is a reagent for introducing protectedsulfhydryls into proteins, peptides and other molecules. They are theN-hydroxysuccinimide (NHS) esters of S-acetylthioacetic and propionicacid. A stable, covalent amide bond was formed from the reaction of theNHS ester with primary amines. The amine was reacted with the NHS esterby nucleophilic attack, with N-hydroxysuccinimide being released as aby-product. Deprotection (deacylation) to generate a sulfhydryl for usein cross-linking and other applications was accomplished usinghydroxylamine.HCl.

The maleimide groups were introduced to the Avidin using Sulfo-SMCC.Sulfo-SMCC is a heterobifunctional cross-linker that contains aN-hydroxysuccinimide (NHS) ester and a maleimide group. NHS esters reactwith primary amines at pH 7-9 to form covalent amide bonds. SMCC andSulfo-SMCC are often used to prepare protein-protein conjugates in atwo-step reaction scheme. First, the amine-containing protein wasreacted with a several-fold molar excess of the cross linker, followedby removal of excess (nonreacted) reagent by desalting or dialysis;finally, the sulfhydryl-containing molecule is added to react with themaleimide groups already attached to the first protein.

The conjugates prepared by cross linking were obtained by reactingmaleimides with sulphydryl groups at pH 6.5-7.5 to form stable thioetherbonds.

An alternative to the procedure above is to use commercially availableMalimide activated Neutravidin instead of the activated Avidin.Maleimide Activated NeutrAvidin™ Protein is for directly preparingNeutrAvidin™ Protein (NAP) conjugates with proteins, peptides, and othermolecules that contain a free sulfhydryl (—SH) group.

Preparation of Dual Affinity Polypeptide (DAP) by Chemical Cross-Linking

Procedure for chemically cross-linking Protein A, and either Avidin orNeutravidin into a conjugate with the required properties of a DAPlinker.

Materials

SATA (N-Succinimidyl S-Acetylthioacetate), (Pierce, no. 26102) D-Salt™Excellulose™ Desalting Column, 5 ml (Pierce No. 20449)

Hydroxylamine.HCl (Pierce, No. 26103), DMSO (Dimethylsulfoxide, Pierce,No. 20688), Sulfo-SMCC: (sulfosuccinimidyl4-(N-maleimidomethyl)cyclohexan-1-carboxylate) (Pierce, 22322), ProteinA (GE Health Care, 17-0872-50), Avidin (Kem-En-Tec, 4020H), Maleimideactivated neutravidin (Pierce, no. 31007), PD-10 Sephadex G-25M (GE;17-0851-01),

HiPrep 26/60 Sephacryl S-100 HR (MW range 1.000-100.000) (GE,17-1194-01)

Anti-IgG Affibody (Affibody, 10.0623.01.0050)

Dithiothreitol ([3483-12-3], Sigma-Aldrich D0632).

Buffers:

PBS Reaction Buffer: 200-500 ml of PBS: 0.1 M phosphate, 0.15 M NaCl, pH7.2

Deacetylation Solution: 0.5 M Hydroxylamine, 25 mM EDTA in PBS, pH 7.2

PBS-EDTA solution: 200-500 ml of PBS: 0.1 M phosphate, 0.15 M NaCl, 5 mMEDTA, pH 7.2.

Phosphate-buffered saline (PBS, pH 7.2; or other amine- and sulfhydrylfree buffer at pH 6.5-7.5 adding EDTA to 1-5 mM helps to chelatedivalent metals, thereby reducing disulfide formation in thesulfhydryl-containing protein.

Procedure for Sulfhydryl Modification of Protein A

A. Reaction of Protein A with SATA (or SATP)

Immediately before reaction, 6.4 mg of SATA was dissolved in 0.5 ml ofDMSO (resulting in ˜55 mM solution).

1.0 ml of Protein A solution (2.6 mg/mL) was then combined with 10 μl ofthe SATA solution. The contents were mixed and incubated at roomtemperature for 30 minutes.

The level of sulfhydryl incorporation may be altered by using differentmolar ratios of SATA to protein. The default reaction uses 60 nmolProtein A and 550 nmol SATA, a 9:1 molar ratio of SATA to protein. Theamount of SATA in the reaction may be increased or decreased by addingmore or less than 10 μl of the SATA solution per ml of Protein Solution.

B. Desalt to Purify Acylated Protein A from Excess Reagent andby-Products

A desalting column was equilibrated with two column volumes of ReactionBuffer. Use at least a 5 ml desalting column for each 1 ml of reactionvolume to be processed.

1.01 ml reaction mixture was applied to the column. Collection of 1 mlfractions was started immediately. After the reaction mixture hadcompletely entered the column bed and the first fraction was collected,at least 10 mL Reaction Buffer was added to the column and collectioncontinued as separate 1 ml fractions as they emerged from the column.

Fraction(s) that contain Protein A were identified as those having peakabsorbance at 280 nm. With a 5 ml desalting column, fractions 2 and 3contained most of the protein, while excess SATA came out in thefollowing fractions. The fractions that contain the modified Protein Awere pooled.

The modified Protein A may be stored indefinitely for laterdeacetylation and generation of sulfhydryl groups (Section C).

C. Deacetylate SATA-Modified Protein A to Generate Sulfhydryl Groups

1.0 ml of SATA-modified (acetylated) Protein A was combined with 100 μlof the Deacetylation Solution. The contents were mixed and incubated 2hours at room temperature.

A desalting column was used to purify the sulfhydryl-modified proteinfrom the Hydroxylamine in the Deacetylation Solution.

Desalting was done into Reaction Buffer containing 10 mM EDTA tominimize disulfide bond formation using the same procedure as in SectionB. Fractions that contained the modified ProteinA were pooled. Theprotein concentration should be ˜1.3 mg/ml. In order to minimizedisulfide formation Section D was performed immediately.

Before or after desalting, the protein may be assayed for sulfhydrylcontent using Ellman's Reagent (Pierce, no 23460 (kit for sulfhydrylgroup detection)).

D. Conjugation of SATA modified Protein A to Maleimide Activated Avidinor Neutravidin.

This method uses approximately equimolar amounts of activated Protein Ato Avidin or Neutravidin.

Example 1a Procedure for Maleimide Modification of Avidin andPreparation of the DAP molecule [Protein A—Avidin]

Generally, a 10- to 50-fold molar excess of cross-linker over the amountof amine-containing protein results in sufficient maleimide activationto enable several sulfhydryl-containing proteins to be conjugated toeach amine-containing protein.

More dilute protein solutions require greater fold molar excess ofreagent to achieve the same activation level. Empirical testing isnecessary to determine optimal activation levels and final conjugationratios for the intended application.

Protocol

For best results, ensure that Protein A-SH is prepared as describedabove and ready to combine with maleimide activated Avidin in step 5.

32 mg Avidin was prepared in 5 mL PBS Buffer, and 4.36 mg sulfo-SMCC wasprepared in 1 mL PBS/EDTA buffer. Then 500 μL of the activation solutionwas transferred to the Avidin solution. The mixture was incubated 30minutes at room temperature. Excess cross-linker was remove using adesalting column equilibrated with PBS-EDTA Buffer.

Protein A-SH and desalted maleimid activated Avidin were combined andmixed in a molar ratio corresponding to approximately 1:1. The reactionmixture was incubated at room temperature overnight.

Generally, there is no harm in allowing the reaction to proceed forseveral hours or overnight, although usually the reaction will becomplete in about 30 min. To stop the conjugation reaction beforecompletion, add buffer containing reduced cysteine at a concentrationseveral times greater than the sulfhydryls of Protein A-SH.

Example 1b Preparation of the DAP Molecule [Protein A—Neutravidin]

For best results, ensure that Protein A-SH is prepared as describedabove and ready to combine with maleimide activated Avidin.

Maleimide activated Neutravidin (Pierce, no 31007) is commerciallyavailable for directly preparing NeutrAvidin™ Protein (NAP) conjugateswith proteins, peptides, and other molecules that contain a freesulfhydryl (—SH) group. NeutrAvidin™ Protein is a modified avidinderivative with several key features that provide a biotin-bindingprotein with exceptionally low non-specific binding properties.NeutrAvidin™ Protein does not contain carbohydrates, renderinglectin-binding activity to undetectable levels. Additionally, theisoelectric point of NAP is 6.3±0.3, which is much lower than nativeAvidin and not as acidic as streptavidin.

Protocol

1.0 mL of ultra pure water was added to suspend 5 mg lyophilizedNeutravidin.

Protein A-SH and maleimid activated Neutravidin were combined and mixedin a molar ratio corresponding to 1:1. The reaction mixture wasincubated at room temperature overnight.

Generally, there is no harm in allowing the reaction to proceed forseveral hours or overnight, although usually the reaction will becomplete in the specified time. To stop the conjugation reaction beforecompletion, add buffer containing reduced cysteine at a concentrationseveral times greater than the sulfhydryls of Protein A-SH.

Example 1c Preparation of the DAP Molecule [Affibody (IgG)—Avidin]Protocol

Avidin (10 mg) was activated with sulfo-SMCC as described in Example 1a.

Anti-IgG Affibody disulfide dimers were reduced to monomers:

Anti-IgG Affibody (5 mg) is dissolved in PBS-buffer (5 mL), and 3.8 mLof this solution is transferred to a vial containing 12.3 mgdithiothreitol (DTT) giving a final DTT concentration of 20 mM solution.This mixture is turned at RT for 2 h.

Upon this, excess DTT is removed by splitting the reaction mixture intwo portions, passing each portion through a PD-10 column (bedvolume 8mL). The columns had been equilibrated with 25 mL PBS buffer before use.The monomeric Anti-IgG Affibody is eluted from the columns in 2×9-10fractions, each containing 1 mL.

By measuring A₂₈₀ of the fractions the protein was located in 2fractions from each column. These fractions were pooled and mixed withthe desalted maleimid activated avidin solution (20 mL) in a molar ratiocorresponding to approximately 1:1 (avidin calculated as monomer;MW=17.000), and the coupling was allowed to proceed overnight at roomtemperature with gently turning of the coupling mixture.

The following day, 1500 μL of the conjugation mixture was concentratedin an Amicon Ultra (cut off 3 kDa) to a total volume of 400 μL, whichwas used for analysis by SDS PAGE. This showed that all avidin hadreacted, and that there was still some unreacted anti-IgG Affibodypresent.

The conjugation mixture was freezed until purified by SEC.

The above protocol can be used for the preparation of all derivatives ofAffibody-Avidin dual affinity polypeptides.

Example 1d Preparation of the DAP Molecule [Affibody (Insulin)—Avidin]

Avidin (9 mg) was activated with sulfo-SMCC as described in Example 1a.

Anti-Insulin Affibody (His₆-Z000810-Cys; P800014) disulfide dimers werereduced to monomers as described in Example 1c.

The pooled fractions from the PD-10 columns were mixed with the desaltedmaleimid activated avidin solution (16.1 mL) in a molar ratiocorresponding to approximately 1:1, and the coupling was allowed toproceed overnight at room temperature with gently turning of thecoupling mixture.

The following day, the conjugation mixture was analyzed by SDS PAGE.This showed that all avidin had reacted, and that there was still someunreacted anti-Insulin Affibody present.

The conjugation mixture was freezed until purified by SEC.

Example 1e Preparation of the DAP Molecule [Affibody (Insulin)—Avidin]

Avidin (9 mg) was activated with sulfo-SMCC as described in Example 1a.

Anti-Insulin Affibody (Insulin, His₆-Z01139-Cys; P800022) disulfidedimers were reduced to monomers as described in Example 3c.

The pooled fractions from the PD-10 columns were mixed with the desaltedmaleimid activated avidin solution (16.1 mL) in a molar ratiocorresponding to approximately 1:1, and the coupling was allowed toproceed overnight at room temperature with gently turning of thecoupling mixture.

The following day, the conjugation mixture was analyzed by SDS PAGE.This showed that all avidin had reacted, and that there was still someunreacted anti-Insulin Affibody present.

The conjugation mixture was freezed until purified by SEC.

Example 2 Recombinant Dual Affinity Constructs for Expression inAspergillus oryzae Strains

Aspergillus oryzae BECh2 is described in WO 00/39322, example 1, whichis further referring to JaL228 described in WO 98/12300, example 1.

JaL1168 is described in example 2 g.

JaL1171 is described in example 2 g.

JaL1174 is described in example 2 g.

JaL1176 is described in example 2 g.

JaL1181 is described in example 2 g.

JaL1210 is described in example 2 g.

MT173 is a derivative of MC1000 (Casadaban & Cohen J. Mol. Biol. 138(1980) 179-207) which are ara⁺ and leuB6.

Genes

AMG: indicate the Aspergillus niger glucoamylase gene (Boel et al. EMBOJournal 3 (1984) 1581-1585)

Z: indicated the Z domain from Staphylococcus aureus protein A (Nilssonet al. Protein Engineering 1 (1987) 107-113).

Pre-CBD_((C315)): indicate the Meripilus giganteus endoglucanase II(DSM971) signal (pre)+cellulose binding domain (CBD)+linker region.

CBD(egv): indicated the Humicola insulens endoglucanase V (DSM1800)linker region+cellulose binding domain.

VhhRR6(2): indicated the variable region from a Llama single chainantibody reacting against the hapten azo-dye Reactive Red (RR6) (Frenkenet al. J. Biotechnology 78 (2000) 11-21.

Plasmids

p775 is described in EP 238023.

pA2C315 is deposited at DSM under the accession no. DSM971. The plasmidcontains a cDNA clone from Meripilus giganteus encoding an endoglucanaseII gene.

pCAMG91 is described in Boel et al. EMBO Journal 3 (1984), 1581-1585.

pJaL790 is described in WO2005070962, example 1.

pJaL1153 is described in example 2c.

pJaL1154 is described in example 2a.

pJaL1158 is described in example 2d.

pJaL1159 is described in example 2a.

pJaL1164 is described in example 2c.

pJaL1165 is described in example 2d.

pJaL1168 is described in example 2e.

pJaL1169 is described in example 2b.

pJaL1170 is described in example 2f.

pJaL1171 is described in example 2b.

pMT2786 is described in WO2006050737 example 2.

pSX320 is described in EP 0 531 372.

Primer and DNA Sequences

Synthetic DNA 1 (SEQ ID NO 1)

Synthetic DNA 2 (SEQ ID NO 4)

Adaptor sequence 1 (SEQ ID NO 5)

Adaptor sequence 2 (SEQ ID NO 6)

primer 8683 (SEQ ID NO 10)

primer CBD:Z-NA (SEQ ID NO 11)

primer Z-NA (SEQ ID NO 12)

primer Z-CA (SEQ ID NO 13)

primer Z-CA:CBD (SEQ ID NO 14)

primer 8654 (SEQ ID NO 15)

Primer CBD:Z-NB (SEQ ID NO 19)

Primer Z-NB (SEQ ID NO 20)

Primer Z-CB (SEQ ID NO 21)

Primer Z-NB:CBD (SEQ ID NO 22)

Methods

General methods of PCR, cloning, ligation nucleotides etc. arewell-known to a person skilled in the art and may for example be foundin “Molecular cloning: A laboratory manual”, Sambrook et al. (1989),Cold Spring Harbor lab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al.(eds.); “Current protocols in Molecular Biology”, John Wiley and Sons,(1995); Harwood, C. R., and Cutting, S. M. (eds.); “DNA Cloning: APractical Approach, Volumes I and II”, D. N. Glover ed. (1985);“Oligonucleotide Synthesis”, M. J. Gait ed. (1984); “Nucleic AcidHybridization”, B. D. Hames & S. J. Higgins eds (1985); “A PracticalGuide To Molecular Cloning”, B. Perbal, (1984).

PCR Amplification

All PCR amplifications were performed in a volume of 100 microLcontaining 2.5 units Tag polymerase, 100 ng of pSO2, 250 nM of eachdNTP, and 10 μmol of each of the two primers described above in areaction buffer of 50 mM KCl, 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl2.Amplification was carried out in a Perkin-Elmer Cetus DNA Termal 480,and consisted of one cycle of 3 minutes at 94° C., followed by 25 cyclesof 1 minute at 94° C., 30 seconds at 55° C., and 1 minute at 72° C.

Aspergillus Transformation

Aspergillus transformation was done as described by Christensen et al.;Biotechnology 1988 6 1419-1422. In short, A. oryzae mycelia were grownin a rich nutrient broth. The mycelia were separated from the broth byfiltration. The enzyme preparation Novozyme® (Novo Nordisk) was added tothe mycelia in osmotically stabilizing buffer such as 1.2 M MgSO₄buffered to pH 5.0 with sodium phosphate. The suspension was incubatedfor 60 minutes at 37 degrees C. with agitation. The protoplast wasfiltered through mira-cloth to remove mycelial debris. The protoplastwas harvested and washed twice with STC (1.2 M sorbitol, 10 mM CaCl₂, 10mM Tris-HCl pH 7.5). The protoplasts were finally re-suspended in200-1000 microl STC.

For transformation, 5 microg DNA was added to 100 microl protoplastsuspension and then 200 microl PEG solution (60% PEG 4000, 10 mM CaCl₂,10 mM Tris-HCl pH 7.5) was added and the mixture is incubated for 20minutes at room temperature. The protoplast were harvested and washedtwice with 1.2 M sorbitol. The protoplast was finally resuspended 200microl 1.2 M sorbitol. Transformants containing an intact niaD gene wereselected for its ability to used nitrate as the sole source for nitrogenon minimal plates (Cove D. J. 1966. Biochem. Biophys. Acta. 113:51-56)containing 1.0 M sucrose as carbon source, 10 mM Sodium nitrate asnitrogen source. After 4-5 days of growth at 37 degrees C., stabletransformants appeared as vigorously growing and sporulating colonies.Transformants were purified twice through conidiospores.

Media and Reagents

YPM medium (2 g/l yeast extract, 2 g/l peptone, and 2% maltose)

Growth of Aspergillus Transformants

Shake flask containing 10 ml YPM medium was inoculated with spores fromthe respective transformants and incubated at 30 degrees C., at 200 rpmfor 4 days.

SDS-Page

SDS gel used was Criterion™ XT precast gels, 10% Bis-Tris, from BIO-RADand was run and stained with Coomassie blue as recommend by themanufactory.

Example 2a Construction of Aspergillus Expression Cassette pJaL1159(Pre-CBD_((C315))-KR::VhhRR6(R2)::Z::Z)

Plasmid pJaL1154 contains a synthetic DNA SEQ ID NO 1 in pUC19 encodinga fusion protein composed of: signal+cellulose binding domain+linkerfrom C315, the amino acids KR, the variable region of a llama singlechain antibody raised against the reactive dye RR6, and a repeat of theZ domain from protein A (pre-CBD_((C315))-KR::VhhRR6(R2)::Z::Z).

Expression vector pJaL1159 was constructed for transcription of thefusion protein pre-CBD_((C315))-KR::VhhRR6(R2)::Z::Z (SEQ ID NO 2). Theplasmid pJaL1154 harboring the fusion protein was digested withBamHI-XhoI. The 966 bp fragment was gel-purified and ligated into theAspergillus expression cassette pMT2786 digested with BamH I-XhoI (a6936 bp fragment). The ligation mixture was transformed into E. coliMT173 using the Saccharomyces cerevisiae Leu2 gene as selective markerto create the expression plasmid pJaL1159. The amplified plasmid wasrecovered using a QIAprep® Spin Miniprep kit (QIAGEN, Chatsworth,Calif.) according to the manufacturer's instructions

Plasmid pMT2786 comprise an expression cassette based on the Aspergillusniger neutral amylase II promoter fused to the Aspergillus nidulanstriose phosphate isomerase non translated leader sequence (Na2/tpipromoter) and the Aspergillus niger amyloglycosidase terminator (AMGterminator), the selective marker amdS from Aspergillus nidulansenabling growth on acetamide as nitrogen source and having theSaccharomyces cerevisiae Leu2 gene for selection in E. coli.

Example 2b

Construction of Aspergillus Expression Cassette pJaL1171Pre-CBD_((C315))KR::VhhRR6(R2)::Z

For construction of the fusion proteinpre-CBD_((C315))-KR::VhhRR6(R2)::Z (SEQ ID NO 3) plasmid pJaL1154 wasdigested with BglII and the 3472 bp fragment was gel-purified andligated with itself resulting in pJaL1169. The 795 bp BamHI-XhoIfragment from pJaL1169 was purified and ligated into the Aspergillusexpression cassette pMT2780 digested with BamH I and XhoI (a 6936 bpfragment). The ligation mixture was transformed into E. coli MT173 usingthe Saccharomyces cerevisiae Leu2 gene as selective marker to create theexpression plasmid pJaL1171. The amplified plasmid was recovered using aQIAprep® Spin Miniprep kit (QIAGEN, Chatsworth, Calif.) according to themanufacturer's instructions.

Example 2c

Construction of Aspergillus Expression Cassette pJaL1164AMG_((1-526aa))::Z::Z

Plasmid pJaL1153 contains a synthetic DNA SEQ ID NO 4 in pUC19 encodinga fusion protein composed of: Aspergillus niger AMG DNA encoding aminoacids 488-526 and a repeat of the Z domain from protein A(AMG_((488-526aa))::Z::Z).

Plasmid pToC100 contains the Aspergillus niger AMG (Boel et al. EMBOJournal 3 (1984), 1581-1585) regulated by the TAKA promoter from p775and at the same time a BamHI site was introduce upfront of the AMG startcodon. pToC100 was constructed by ligating the following fragmentstogether: 4306 bp BamHI-NcoI fragment from p775, an adapter SEQ ID NO: 5and SEQ ID NO.: 6, 860 bp BssHII-Pst1 from pCAMG91 and 1410 bp PstI-NcoIfragment from pCAMG91.

Expression vector pJaL1164 was constructed for transcription of thefusion protein AMG_((1-526aa))::Z::Z (SEQ ID NO 7). A 1723 bpBamHI-DraIII fragment and a 458 bp DraIII-XhoI fragment was gel-purifiedfrom plasmid pToC100 and pJaL1153, respectively, and ligated into theAspergillus expression cassette pMT2786 digested with BamH 1-XhoI (a6936 bp fragment). The ligation mixture was transformed into E. coliMT173 using the Saccharomyces cerevisiae Leu2 gene as selective markerto create the expression plasmid pJaL1164. The amplified plasmid wasrecovered using a QIAprep® Spin Miniprep kit (QIAGEN, Chatsworth,Calif.) according to the manufacturer's instructions.

Example 2d Construction of Aspergillus Expression Cassette pJaL1165AMG_((1-526aa))::Z

For construction of an expression plasmid encoding for the fusionprotein AMG_((1-526aa))::Z (SEQ ID NO 8) plasmid pJaL1153 was digestedwith BglII and the 2969 bp fragment was gel-purified and ligated withitself resulting in pJaL1158. A 1723 bp BamHI-DraIII fragment frompToC100 and a 287 bp fragment from pJaL1158 was purified and ligatedinto the Aspergillus expression cassette pMT2786 digested with BamH Iand XhoI (a 6936 bp fragment). The ligation mixture was transformed intoE. coli MT173 using the Saccharomyces cerevisiae Leu2 gene as selectivemarker to create the expression plasmid pJaL1165. The amplified plasmidwas recovered using a QIAprep® Spin Miniprep kit (QIAGEN, Chatsworth,Calif.) according to the manufacturer's instructions.

Example 2e Construction of Aspergillus Expression Cassette pJaL1168Pre-CBD_((C315))::Z::Z::CBD_((egv))

Construction of the expression plasmid pJaL1168 encoding for the fusionprotein pre-CBD_((C315))::Z::Z::CBD_((EGV)) (SEQ ID NO 9) was done byamplification by PCR: 1) of the pre-CBD_((C315)) region using pA2C315 astemplate and the primer pair 8683/CBD:Z-NA (SEQ ID NO 10 and 11), 2) ofthe Z::Z region using pJaL1153 as template and the primer pair Z-NA/Z-CA(SEQ ID NO 12 and 13) and 3) of the CBD_((EGV)) region using pSX320 astemplate and the primer pair Z-CA:CBD/8654 (SEQ ID NO 14 and 15),resulting in 3 DNA fragments of 337 bp, 382 bp and 344 bp, respectively.The 3 fragments were mixed and used as template for amplification by PCRwith primer pair 8653/8654 of a 983 bp fragment. The PCR fragment wasdigested with BamHI-HindIII and the 798 bp fragment was purified andclone ligated into the Aspergillus expression cassette pJaL790 digestedwith BamH I-HindIII (a 7386 bp fragment). The ligation mixture wastransformed into E. coli DB6507 using the Saccharomyces cerevisiae Ura3gene as selective marker to create the expression plasmid pJaL1168. Theamplified plasmid was recovered using a QIAprep® Spin Miniprep kit(QIAGEN, Chatsworth, Calif.) according to the manufacturer'sinstructions.

Plasmid pJaL790 comprised an expression cassette based on theAspergillus niger neutral amylase II promoter fused to the Aspergillusnidulans triose phosphate isomerase non translated leader sequence(Na2/tpi promoter) and the Aspergillus niger amyloglycosidase terminator(AMG terminator), the selective marker amdS from Aspergillus oryzaeenabling growth on acetamide as nitrogen source.

Example 2f Construction of Aspergillus Expression Cassette pJaL1170Pre-CBD_((C315))-KR::Z::Z::CBD_((C315))::CBD_((EGV))

Plasmid pJaL802 is an Aspergillus expression plasmid builds on pJaL790which contains a DNA (SEQ ID NO 16) encoding for the fusion proteinpre-CBD_((C315))::CBD_((EGV)) (SEQ ID NO 17).

Construction of the expression plasmid pJaL1170 encoding for the fusionprotein pre-CBD_((C315))-KR::Z::Z::CBD_((C315))::CBD_((EGV)) (SEQ ID NO18) was done by amplification by PCR: 1) of the pre-CBD_((C315))-KRregion using pA2C315 as template and the primer pair 8683/CBD:Z-NB (SEQID NO 10 and 19), 2) of the Z::Z region using pJaL1153 as template andthe primer pair Z-NB/Z-CB (SEQ ID NO 20 and 21) and 3) of theCBD_((C315))-CBD_((EGV)) region using pJaL802 as template and the primerpair Z-CB:CBD/8654 (SEQ ID NO 22 and 15), resulting in 3 DNA fragmentsof 343 bp, 382 bp and 443 bp, respectively. The 3 fragments were mixedand used as template for amplification by PCR with primer pair 8653/8654of a 1088 bp fragment. The PCR fragment was digested with BamHI-HindIIIand the 894 bp fragment was purified and clone ligated into theAspergillus expression cassette pJaL790 digested with BamH I-HindIII (a7386 bp fragment). The ligation mixture was transformed into E. coliDB6507 using the Saccharomyces cerevisiae Ura3 gene as selective markerto create the expression plasmid pJaL1170. The amplified plasmid wasrecovered using a QIAprep® Spin Miniprep kit (QIAGEN, Chatsworth,Calif.) according to the manufacturer's instructions,

Example 2g Expression of DAP in Aspergillus oryzae Strains

The Aspergillus oryzae strains BECh2 was transformed with the expressionplasmid pJaL1159, pJaL1164, pJaL1165, pJaL1168, pJaL1170 and pJaL1171 asdescribed under methods.

A shake flask containing 10 ml YPM medium (2 g/l yeast extract, 2 g/lpeptone, and 2% maltose) was inoculated with spores from the generatedtransformants and the host BECh2 and incubated at 30° C., with shaking(200 rpm) for 4 days. Supernatants (10 μl) were analysed on SDS-page. Atransformant producing the desired protein from each plasmid pJaL1159,pJaL1164, pJaL1165, pJaL1168, pJaL1170 and pJaL1171 was named JaL1210,JaL1168, JaL1171, JaL1174, JaL1176 and JaL1181, respectively. Productsof the expected size from each transformant were confirmed by SDS-page.The amino acids sequence of each construct produced in JaL1210(VhhRR6(R2)::Z::Z), JaL1168 (AMG_((1-526aa))::Z::Z), JaL1171(AMG_((1-526aa))::Z), JaL1174 (CBD_((C315))::Z::Z::CBD_((egv))), JaL1176(Z::Z::CBD_((C315)):: CBD_((egv))) and JaL1181 (VhhRR6(R2)::Z) are shownin SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27and SEQ ID NO 28, respectively.

Example 3 Recombinant Dual Affinity Constructs for Expression inBacillus licheniformis Media

LB agar, TY buillon medium and BPX shake flask medium have all beendescribed in Patent Publication WO 94/14968.

CAL 18-2 media (11): Yeast extract (#0127-17-9 Difco Laboratories, MI,USA) 40 g; Magnesium Sulfate (#5886 Merck, Darmstadt, Germany) 1.3 g;Glucidex 12 (Roquette Feres, France) 50 g; Sodium Di-hydrogenphosphate(#6346 Merck, Darmstadt, Germany) 20 g; EDF-Tracemetals (recipe seebelow) 6.7 ml; Na₂MoO₄-Tracemetals (recipe see below) 6.7 ml; PluronicPE6100 (BASF, Germany) 0.1 ml; Ionexchanged water adjust to 1000 ml. Allis mixed, volume is adjusted, pH is measured and adjusted to pH 6.0using NaOH. The media is sterilised by aotoclaving at 121° C. for 20min. EDF-Tracemetals (11): Mangan (II) sulphate (#5963 Merck, Darmstadt,Germany) 4.48 g; Iron (III) chloride (#3943 Merck, Darmstadt, Germany)3.33 g; Copper (II) sulphate (#2790 Merck, Darmstadt, Germany) 0.625 g;Zinksulphate (#8883 Merck, Darmstadt, Germany) 7.12 g; Ionexchangedwater adjust to 1000 ml. All is mixed, volume is adjusted. Solution isfilter-sterilized and kept at 4° C. Na₂MoO₄-Tracemetals (11):SodiumMolybdat (#6521 Merck, Darmstadt, Germany) 2.0 g; Ionexchangedwater adjust to 1000 ml. All is mixed, volume is adjusted. Solution isfiltersterilized and kept at 4° C.

Strains and Donor Organisms

Bacillus subtilis PL1801. This strain is the B. subtilis DN1885 withdisrupted apr and npr genes (Diderichsen, B., Wedsted, U., Hedegaard,L., Jensen, B. R., Sjoholm, C. (1990) Cloning of aldB, which encodesalpha-acetolactate decarboxylase, an exoenzyme from i Bacillus brevis.J. Bacteriol., 172, 4315-4321).

Bacillus subtilis PP289-5. This strain is a donor strain for conjugationof plasmids to Bacillus licheniformis described in U.S. Pat. No.5,843,720

Bacillus licheniformis MDT223 described in patent WO2005/123915

Genes

Z: indicate the Z domain from Staphylococcus aureus protein A (Nilssonet al. Protein Engineering 1 (1987) 107-113). The Z gene is a syntheticgene assembled by primers (SEQ ID NO 29)

Streptavidin gene: indicate the gene encoding Streptavidin fromStreptomyces avidinii as described by Argarana (Argarana et. Al. (1986)Nucleic Acids Res. 14, 1871-1882). The gene encoding streptavidin is asynthetic gene (SEQ ID NO 30)

A synthetic gene can be constructed by PCR assembly of overlappingoligonucleotides in various methods described eg. by Stemmer et al, Gene164, pp-49-53, 1995; Dillon and Rossen, BioTechniques 9, 298-300, 1990;Prodromou and Pearl, Protein Engineering 5, 827-829, 1992; Chn et al.,Journal of Amarican Chemical Society 11, 8799-8800, 1994 and others.Such genes may also simply be purchased through one of many commercialcompanies.

Plasmids

pSJ6208 is an E. coli pUC derivative described in SEQ ID NO 31.

pSJ6321 is a pE194 derivative with an erythromycin marker gene. Theplasmid also holds cryIIIA stabilizer sequence, DNA encoding the signalpeptide of amyL fused to a protease followed by a downstream sequence ofamyL (SEQ ID NO 32).

pMOL2743 is described in this example (SEQ ID NO 33)

pMOL2744 is described in this example (SEQ ID NO 34)

pMOL2746b is described in this example (SEQ ID NO 35)

Construction of Integration Vector Z::Z::streptavidin for B.licheniformis Expression

The synthetic gene (SEQ ID NO 29) encoding the Z::Z domaine wasamplified by the primers SEQ ID NO 36 and SEQ ID NO 37:

SEQ ID NO 36: TCATTCTGCAGCAGCGGCGGATAACAAATTTAACAAAGAACAGCA-GAACGCGTTTTATGAAA SEQ ID NO 37:AACTAAGCGGCCGCTAGCGACTACACTTTAGGAGCTTGCGCGTCAT- TAAGCT

The PCR fragment was digested with PstI-EagI and the 368 bp fragment waspurified and ligated into the E. coli pUC derivative plasmid pSJ6208(SEQ ID NO 31) digested with PstI EagI giving a 3389 bp fragment. Theligation mixture was transformed into E. coli SJ2 (Diderichsen, B.,Wedsted, U., Hedegaard, L., Jensen, B. R., Sjoholm, C. (1990). Cloningof aldB, which encodes acetolactate decarboxylase, an exoenzyme fromBacillus brevis). The plasmid holding the sub cloned Z::Z gene pMOL2743(SEQ ID NO 33), was recovered using a QIAprep® Spin Miniprep kit(QIAGEN, Chatsworth, Calif.) according to the manufacturer'sinstructions.

The synthetic gene (SEQ ID NO 30) encoding streptavidin was digestedwith HindIII giving a fragment of 620 bp. The plasmid pMOL2743 wasrestricted with HindIII and treated with alcaline phosphatase to avoidrelegation. The two fragments were ligated and transformed to the E.coli SJ2. The colonies were screened for presence of the streptavidingene and clones were picked where the streptavidin gene is inserted inthe right orientation giving rise to the plasmid pMOL2744 (SEQ ID NO34). In this plasmid the genes encoding the Z::Z domaine andStreptavidin if translationally fused.

The hybrid gene encoding Z::Z::Streptavidin was transferred to anintegration vector designed to allow integration of the amylaseexpression cassette into the chromosome of a B. licheniformis strain,that already contains an artificial tandem promoter integrated at theamyL locus, as described in example 6 of WO2005/123915. This wasachieved by a ligation of three fragments. The first fragment is aPstI-BglII restriction digest of pMOL2744 giving rise to a 931 bpfragment. The second fragment is a BgIII-BamHI restriction digest of theplasmid pSJ6321 (SEQ ID NO 32) isolating a 4288 bp fragment. The thirdfragment is a BamHI-PstI digest of pSJ 6321 isolating a 1234 bpfragment. The three fragments were ligated and introduced bytransformation into PL1801 giving rise to an integration vectorpMOL2746b (SEQ ID NO 35).

This pMOL2746b plasmid is then re-transformed by either competence,electroporation or conjugation into a protease deficient Bacilluslicheniformis and inserted by double homologoes integration at the amyLlocus using an already inserted cryIIIA sequence and the amyl downstreamsequence. The resulting Bacillus licheniformis strain has the artificialtandem promoter and cryIIIA sequence driving the Z::Z::Strepavidinexpression from the amyL locus. The Bacillus licheniformis host ispreferred to be protease deficient to allow expression of theZ::Z::Streptavidin hybrid protein. The following proteases can bedeleted by standard techniques using double homologous recombination:mpr, aprE, nprE, vpr, bpr, epr, wprA and ispA.

The protease deficient Bacillus licheniformis host with the expressioncassette encoding the Z::Z::Strepatavidin hybrid DAP protein isfermented in 100 ml shake flasks with CAL18-2 medium described above at30° C., 300 rpm for 2 days. Samples are taken out day one and day two toevaluate the DAP expression on an SDS gel. The data show a protein bandat the right size of 30 KDa.

The PCR fragment is digested with the restriction enzymes Rsa I and HindIII and the resulting 489 bp fragment is cloned into the 5327 pStrExp1digested with the restriction enzymes Nru I and Hind III by DNA ligationand cloning into B. subtilis PL1801.

The DNA sequence of the open reading frame encoding the fusion proteinis shown in SEQ ID NO: 38, and the protein sequence in SEQ ID NO: 39.

Example 4 Expression of the DAP Construct in Bacillus subtilis

Transformants of B. Subtilis PL1801 is grown in shake flasks asdescribed in patent WO 2000/075344 and the fusion protein is recoveredfrom the supernatant. The antibody binding and biotin binding propertiesof the fusion protein is confirmed as shown elsewhere herein.

Example 5 Purification of DAP

5a. Chemically Synthesized DAP Purified by Size Exclusion Chromatography

The conjugation mixtures from example 1 were loaded on a size exclusioncolumn in order to purify the DAP-molecules by removing reactants. Thesize exclusion chromatography was performed on a prepacked Superdex 20016/60 column. 1 mL of DAP reaction solution was loaded on the column.The pump flow was 1.00 mL/min, the eluent was 150 mM NaCl, 50 mM HepespH 7.0 and fractions of 1 mL were collected. Fractions were pooled basedon absorbance measurements at 280 nm in order to remove reactants.

The collected and pooled fractions were concentrated 10× using AmiconUltracentrifuge tubes with a NWCO of 3000.

5b. Recombinant DAP by IgG-Sepharose Affinity Chromatography

Recombinant DAP molecules were purified from the fermentation broth bysterile filtration and subsequent column purification by IgG affinitychromatography.

The IgG-sepharose column was prepared by following the proceduresupplied by the vendor:

15 g CNBr-activated sepharose 4B from GE Healthcare was washed for 15min with 3 L1 mM HCl. The washed medium was added to 25 mL 20 mg/mL IgGsolution from DAKO NSA (X0903) and 50 mL 0.75 M NaCl, 0.15 M NaHCO₃ pH8.3. The mixture was gently rotated for 95 min. at room temperature.Excess IgG was washed away with 75 mL 0.1 M NaHCO₃ pH 8.3 containing 0.5M NaCl before the medium was incubated in 0.1 M Tris/HCl pH 8.0 for 2hours. The medium was stored in 20% ethanol until use.

Generic Procedure for Purification of Recombinant DAP

The recombinant DAP molecules from examples 2 and 3 were purified by ageneric affinity chromatographic method that takes advantage of the IgGbinding domain shared by all the recombinant DAP constructs. The DAPmolecules were purified from the sterile filtered fermentation broth.

The chromatography was performed on a XK26/20 column packed with approx.30 mL IgG-sepharose. The fermentation broths were sterile filtered andbetween 65 mL and 80 mL was loaded depending on the volume of thefiltered fermentation broth. The pump flow was 1.50 mL/min during sampleload and 2 mL/min during wash and elution. Buffer A was 0.1 M NaH₂PO₄ pH7.2, 0.15 M NaCl and buffer B: 0.1 M Citric acid pH 3.5. The column waswashed with 15 column volumes buffer A following the sample load. Thebound material was eluted with 5 column volumes buffer B before thecolumn was regenerated with 10 column volumes buffer A. Fractions of 10mL were collected. Eluate having an increase of absorbance at 280 nm waspooled and the pH was adjusted to 7.2 using 1 M Tris. The concentrationof the DAP protein was calculated from the absorption at 280 nm and thetheoretical absorption coefficients calculated from the primary sequenceusing GPMAW 8.0 (Trends in Biochemical Science, Vol 26, No. 11, November2001, pp 687-689, “GPMAW—a software tool for analyzing proteins andpeptides”; see also http://www.qpmaw.comi). The Mw of the purifiedproteins were determined by SDS-PAGE. The samples were stored at −18° C.before further analysis.

Example 6 Characterization of DAP Molecules with Regard to BindingStrength Measured on the Biacore Instrument

The commercialised surface plasmon resonance (SPR) technology for realtime monitoring biomolecular binding event is used to measure thebinding affinities for the prepared DAP candidates. The generalprinciple of this technology is that a SPR sensor chip measures changesin refractive index, and the changes in refractive index correlate tochanges in mass in the aqueous layer close to the sensor surface. Whentarget molecules in solution bind to ligands, immobilised on the sensorsurface, the mass increases, and when they dissociate from the ligandsthe mass decreases. This principle facilitates a continuous, real timemonitoring of the association and dissociation of interacting molecules.The graphical presentation of the relationship provides quantitativeinformation in real time on the binding specificity, activeconcentration of molecule in a sample, reaction kinetics and affinity.

In order to evaluate the binding affinities for both of the bindingaffinities present in the DAP either IgG or the capturing ligand had tobe immobilized onto the sensor chip. For IgG rabbit anti-Mannanase wasused and for the ligand the chip was coated with streptavidin and theligand was then immobilized via biotin linked to the appropriate ligand.

In order to measure the binding to RR6 and acarbose the followingcompounds were prepared; biotin-linked acarbose and biotin-linked RR6.

Preparation of Biotin Linked Acarbose

Biotin (5 mg, 26 μmol) was dissolved in DMF (250 μL) in a 2 mL eppendorftube, and to this EDC.HCl (5 mg, 26 μmol) was added. The mixture wasstirred at RT for 30 min. Acarbose (16.1 mg, 25 μmol) was dissolved inDMF (250 μL) in a 2 mL eppendorf tube, and to this the activated biotinsolution was added dropwise, while the reaction mixture was gentlystirred. When all activated biotin solution was added, 100 μL DMF wasused to wash the reaction container, and these 100 μL were also added tothe acarbose solution. The reaction mixture was left stirring for 2 h atRT. Upon this, the DMF was removed by freeze-drying overnight at −5° C.The crude product was stored at −18° C., until used in the Biacoreexperiments.

Preparation of biotin linked Reactive Red 6

Biotin (5 mg, 26 μmol) was dissolved in DMF (250 μL) in a 2 mL eppendorftube, and to this EDC.HCl (5 mg, 26 μmol) was added. The mixture wasstirred at RT for 30 min. 1,4-Diaminobutane (25.1 μL, 25 μmol) wasdissolved in DMF (250 μL) in a 2 mL eppendorf tube, and to this theactivated biotin solution was added dropwise, while the reaction mixturewas gently stirred. When all activated biotin solution was added, 100 μLDMF was used to wash the reaction container, and these 100 μL were alsoadded to the diaminobutane solution. The reaction mixture was leftstirring for 2 h at RT.

Reactive Red 6 (24.4 mg, 25 μmol) was dissolved in DMF (250 μL) in a 2mL eppendorf tube, and to this the biotin-amide solution was addeddropwise, while the reaction mixture was gently stirred. When allbiotin-amide solution was added, 100 μl DMF was used to wash thereaction container and these 100 μL were also added to the RR6 solution.

The reaction mixture was left stirring overnight at RT. The DMF wasremoved by freeze-drying overnight at −5° C. The crude product wasstored at −18° C., until used in the Biacore experiments.

Biacore Evaluation:

The DAP candidates were analysed for binding to the capturing ligandsensor chip and the target biomolecule sensor chip respectively. ABiacore 3000 instrument was used.

To study the interactions between the IgG-binding end of the DAPmolecule and IgG on the one hand, and the interaction between theligand-binding end and the ligand (biotin, acarbose, reactive red) onthe other hand, IgG and ligand were immobilized onto the sensor surfaceof a sensor chip as described below.

Immobilisation occurred by direct covalent coupling to the surface(using the Amine Coupling Kit, BiaCore, GE Health Care) or via acapturing molecule as prescribed by the manufacturer (BiaCore, GE HealthCare). The amount of coupled target was quantified and expressed inRefractive Units (RU).

Interactions were monitored by injecting samples (20 μl/min) over theprepared sensor surface of the chip. Unless stated otherwise, thebinding was assessed in 10 mM sodium acetatbuffer pH 5.0 at roomtemperature.

Experiment 1 Chip: CM5

Immobilized target: rabbit anti-Mannanase (10 pg/ml) in 10 mMacetatbuffer pH 5.0.

Target RU: 1250 RU

Actual RU:

FC1: 1349

FC2: 1492

FC3: 1338

FC4: 1331

Samples:

FC1: Protein A, 1 pg/ml injected

FC2: Protein A—Avidin, 0.5 pg/ml injected

FC3: Protein A—Avidin, 0.5 pg/ml injected

FC4: Affibody (IgG)—Avidin, 0.6 pg/ml injected

Results:

ka kd KA KD Final RU Protein A Chi₂ = 0.06 4.35e5  3.4e−6 1.28e117.81e−12 140 Protein A- Avidin Chi₂ = 0.737 6.99e5 1.75e−7 3.99e12 2.5e−13 130 Chi₂ = 1.82 1.21e6 5.03e−5 2.41e10 4.15e−11 130Affibody(IgG)- Avidin Chi₂ = 0.505 9.56e4 6.41e−8 1.49e12 6.7e−13 105

Experiment 2 Chip: CM5

Immobilized target: rabbit anti-Mannanase (10 μg/ml) in 10 mMacetatbuffer pH 5.0.

Target RU: 1250 RU

Actual RU:

FC2: 1697

FC3: 1665

Samples:

FC2: CBD-Z-Z-CBD, 10 μg/ml injected

FC3: CBD-Z-Z-CBD, 10 μg/ml injected

Results:

CBD-Z-Z- CBD ka kd KA KD Final RU Chi₂ = 2.3 1.46e5   2e−4  7.3e81.37e−9 200 Chi₂ = 3.78 1.23e5 2.08e−4 5.93e8 1.69e−9 200

Experiment 3 Chip: CM5

Immobilized target: rabbit anti-Mannanase (10 μg/ml) in 10 mMacetatbuffer pH 5.0.

Target RU: 1250 RU

Actual RU:

FC3: 1485

FC4: 1760

Samples:

FC3: AMG-Z, 10 pg/ml injected

FC4: AMG-ZZ, 10 pg/ml injected

Results:

ka kd KA KD Final RU AMG-Z Chi₂ = 6.76 2.69e6 1.08e−2 2.49e8 4.02e−9 5AMG-ZZ Chi₂ = 2.97 3.46e5  4.1e−4 8.42e8 1.19e−9 150

Experiment 4 Chip: CM5

Immobilized target: rabbit anti-Mannanase (1 pg/ml) in 10 mMacetatbuffer pH 5.0.

Target RU: 625 RU

Actual RU:

FC1: 683

FC2: 731

FC3: 881

FC4: 716

Samples:

FC1: Protein A, 1 pg/ml injected

FC2: ZZ-CBD-CBD, 0.1 pg/ml injected

FC3: VhhRR6(R2)-Z, 1 pg/ml injected

FC4: CBD-Z-Z-CBD, 1 pg/ml injected

Results:

ka kd KA KD Final RU Protein A Chi₂ = 1.22 4.27e5 1.26e−4 3.38e92.95e−10 55 ZZ-CBD-CBD Chi₂ = 0.116  4.6e6  3.3e−4  1.39e10 7.18e−11 70VhhRR6(R2)-Z Chi₂ = 2.35  3.2e5   1e−3  3.2e8 3.13e−9  70 CBD-Z-Z-CBDChi₂ = 1.96 2.65e5 2.97e−4 8.94e8 1.12e−9  50

Experiment 5 Chip: SA

Immobilization: None. The Chip is pre-coated with Streptavidin.

Immobilisation of the ligand through Biotin-Streptavidin binding

FC1: Biotin-Acarbose, 10 μg/ml

FC2: Biotin-Acarbose, 10 μg/ml

FC3: Biotin-Reactive Red 6, 10 μg/ml

Binding of the DAP via the ligand-binding end of the molecule to theligand.

FC1: AMG-Z-Z, 10 μg/ml

FC2: AMG-Z, 10 μg/ml

FC3: VhhRR6(R2)-Z, 10 μg/ml

Results:

ka kd KA KD Final RU AMG-Z-Z Chi₂ = 1.38 6.29e3 2.69e−3 2.34e6 4.27e−715 AMG-Z Chi₂ = 2.9 1.02e4  1.8e−3 5.64e6 1.77e−7 20 VhhRR6(R2)-Z Chi₂ =3.24 1.72e4 3.99e−3 4.31e6 2.32e−7 15

Experiment 6 Chip: SA

Immobilization: None. The Chip is pre-coated with Streptavidin.

Immobilisation of the ligand through Biotin-Streptavidin binding

FC4: Biotin-Reactive Red 6, 10 μg/ml

Binding of the DAP via the ligand-binding end of the molecule to theligand in the presence of IgG binding to the Z-domain of the DAPmolecule.

FC4: VhhRR6(R2)-Z+IgG 9.3 μg/ml

Result:

VhhRR6(R2)- Z + IgG ka kd KA KD Final RU Chi₂ = 5.3 225 6.18e−3 3.65e42.74e−5 5

TABLE 1 IgG binding Ligand binding k_(a) k_(d) K_(D) k_(a) k_(d) K_(D)DAP (1/Ms) (1/s) (M) Ligand (1/Ms) (1/s) (M) Avidin-Protein A 1 × 10⁶ 3× 10⁻⁵ 2 × 10⁻¹¹ Biotin^(a) — — — Avidin-Affibody (IgG) 1 × 10³ 6 × 10⁻⁸7 × 10⁻¹³ Biotin — — — AMG-ZZ 4 × 10⁵ 4 × 10⁻⁴ 1 × 10⁻⁹  Acarbose 6 ×10³ 3 × 10⁻³ 4 × 10⁻⁷ AMG-Z 3 × 10⁶ 1 × 10⁻² 4 × 10⁻⁹  Acarbose 1 × 10⁴2 × 10⁻³ 2 × 10⁻⁷ CBD-ZZ-CBD 2 × 10⁵ 2 × 10⁻⁴ 1 × 10⁻⁹  CelluloseZZ-CBD-CBD 5 × 10⁶ 3 × 10⁻⁴ 7 × 10⁻¹¹ Cellulose VhhRR6(R2)-Z 3 × 10⁵ 1 ×10⁻³ 3 × 10⁻⁹  RR6^(b) 2 × 10⁴ 4 × 10⁻³ 2 × 10⁻⁷ Protein A control 5 ×10⁵ 7 × 10⁻⁵ 2 × 10⁻¹⁰ — — — — ^(a)Binding between biotin andavidin/streptavidin is not measured, since it is known to be very tight,and no dissociation can therefore be measured. ^(b)If VhhRR6(R2)-Z ismixed with IgG before loading to the Biacore, the K_(D) is 10⁻⁵M,however, the binding is broken completely as soon as the injection isstopped.

Example 7 Purification of Antibodies Using Dual Affinity PolypeptidePurification Technology

Below is a short description of a generally applicable procedure forimmunoglobulin purification:

-   -   1. A disposable generic solid phase with a low molecular weight        capturing ligand molecule.    -   2. The dual affinity polypeptide DAP (e.g. Avidin-Protein A)        reacts with the IgG (target biomolecule) in the solution. The        DAP molecules are immobilized on the solid phase (e.g.        biotin-agarose) in a complex together with the target protein        (IgG).    -   3. The column is washed to eliminate interfering non-product        components.    -   4. The immunoglobulin is eluted from the column using an        appropriate buffer at low pH.    -   5. The antibody containing fractions are collected and pH is        neutralized.    -   6. The generic matrix containing immobilized dual affinity        polypeptide may be discarded.

Materials and Methods:

All buffer ingredients were pro-analysis. Rabbit serum proteins andRabbit IgG fraction, code X0903 were from DAKO NS, Denmark. BiotinAgarose was from Sigma-Aldrich; B6885-5mL.

The purification experiments were performed using a chromatographysystem comprising a fraction collector (Frac-100), a recorder (Rec-1),an optical unit and a control UV-1, all from GE Healthcare. For allexperiments we used a BioRAd Econo-column ID 1.0 cm with a flow adaptor.The equilibration and dilution buffer was 0.1 M phosphate, 0.15 M NaCl,pH=7.2 (PBS). The elution buffer was 0.1 M citrate, pH=3.5, oralternatively 0.1 M glycine, pH=2.8.

The data processing was based on Abs 280 nm measurement using aPharmacia Gene Quant II and the extinction coefficient for Rabbit IgG (1g/L) of 1.35.

IgG Purification Analysis

All experiments were performed at room temperature.

Example 7a Recovery of Rabbit IgG with free DAP [Protein A—Neutravidin]

The sorbent (Biotin Agarose, 5 mL) was allowed to settle in the columnfor 10 min. The column was packed at a flow of 1.46 mL/min. The columnwas equilibrated with 7.5 column volumes (CV) PBS. DAP (ProteinA-Neutravidin) solution (4.5 mL from example 1b) was mixed with 100 μLRabbit IgG stock solution (20 g/L) and incubated for 5 minutes on amagnetic stirrer. The reaction solution was loaded on the column andwashed with 7.5 CV of PBS to remove excess of target protein. The IgGwas recovered by elution with 3 CV of 0.1 M glycine buffer (pH 2.8). 5mL fractions were collected and analyzed for IgG content by Abs 280. SeeTable 2 for results.

Example 7b Recovery of Rabbit IgG with Immobilized DAP [ProteinA—Neutravidin]

The gel material from example 7a with immobilized DAP [ProteinA—Neutravidin] was regenerated with 7.5 CV PBS before analyzing theconventional affinity purification capabilities.

We loaded 2 mg IgG in 4.6 mL PBS solution on the column. After sampleloading, the column was washed with 7.5 CV of PBS to remove excess ofprotein. Then the column was eluted with 3 CV of 0.1 M Glycine buffer(pH 2.8) to recover the IgG. 5 mL fractions were collected and analyzedfor IgG content by Abs 280 and SDS-PAGE. See Table 2 for results.

TABLE 2 Flow through Recovered Example 1b - Free DAP 74% 26% Example1b - Immobilized DAP 86% 14%

As seen in Table 2, we obtained approximately twice the bindingcapability (26% versus 14%) when DAP and IgG are reacted in solutionprior to loading on the column compared to the conventional affinitychromatography applying immobilized DAP.

Example 7c Purification of Rabbit IgG from Serum with free DAP [ProteinA—Neutravidin]

We studied purification of IgG from rabbit serum to show the specificityof the DAP purification technology.

Approximately 1 mL of sorbent (Biotin Agarose) was allowed to settle inthe column for 10 min. The column was packed at a flow of 1.46 mL/min.The column was equilibrated with 7.5 column volumes (CV) PBS. A 4.5 mLDAP (Protein A—Neutravidin) solution (from example 1b) were mixed with115 μL rabbit serum and incubated for 5 minutes on a magnetic stirrer.The reaction solution was loaded on the column following a wash with 7.5CV of PBS to remove excess of target protein. The IgG was recovered byelution with 3 CV of glycine buffer. 2.5 mL fractions were collected andanalyzed for IgG content by Abs 280 and SDS-PAGE. See Table 3 forresults.

Example 7d Purification of Rabbit IgG from Serum with Immobilized DAP[Protein A—Neutravidin]

The gel from example 1c with immobilized DAP [Protein A—Neutravidin] wasregenerated with 7.5 CV PBS before analyzing the conventional affinitypurification capabilities

We loaded rabbit serum (115 μL rabbit serum in 4.5 mL PBS) solution onthe column. After sample loading, the column was washed with 7.5 CV ofPBS to remove excess of protein. Then the column was eluted with 3 CV of0.1 M Glycine buffer (pH 2.8) to recover the target IgG. The gel wasregenerated with 7.5 CV PBS before the next affinity purification cycle.2.5 mL fractions were collected and analyzed for IgG content by Abs 280and SDS-PAGE. See Table 3 for results.

TABLE 3 Recovered IgG (mg) from serum Example 1b - Free DAP 0.31 Example1b - Immobilised DAP 0.18

As seen in Table 3, we obtained approximately twice the bindingcapability (0.31 mg versus 0.18 mg IgG), when DAP was reacted withrabbit serum in solution prior to contacting the biotin-agarose columncompared to the conventional affinity chromatography applyingimmobilized DAP.

SDS-PAGE showed that only IgG molecules were obtained from serum,showing that the DAP purification technology is specific.

We repeated the analysis using the other DAP conjugate (ProteinA-Avidin) and performed similar tests as above but included an analysisof the leakage of DAP from the column by repeated binding analysis tothe immobilized DAP.

Example 7e Recovery of Rabbit IgG with free DAP [Protein A—Avidin]

The sorbent (Biotin Agarose, 1 mL) was allowed to settle in the columnfor 10 min. The column was packed at a flow of 1.46 mL/min. The columnwas equilibrated with 7.5 column volumes (CV) PBS. 2 mL of DAP (ProteinA—Avidin) solution (from example 3a) was mixed with 160 μL Rabbit IgGstock solution (20 g/L) and incubated for 5 minutes on a magneticstirrer. The reaction solution (˜2 CV) was loaded on the columnfollowing a wash with 7.5 CV of PBS to remove excess of target protein.The IgG was recovered by elution with 3 CV of 0.1 M Citrate, pH=3.5. 2.5mL fractions were collected and analyzed for IgG content by Abs 280. Theresults are shown in Table 4.

TABLE 4 Free DAP versus reuses of immobilised DAP technology (ProteinA - Avidin conjugate) Rabbit IgG recovery 1. reuse of 2. reuse of 3.reuse of 4. reuse of Free immobilsed immobilsed immobilsed immobilsedDAP DAP DAP DAP DAP IgG Re- 1.20 0.30 0.40 0.40 0.30 covered (mg)

Example 7f Recovery of Rabbit IgG with Immobilized DAP [ProteinA—Avidin]

The gel from example 7e with immobilized DAP [Protein A—Avidin] wasregenerated with 7.5 CV PBS before analyzing the conventional affinitypurification capabilities The rabbit IgG stock solution (20 g/L) wasdiluted to a concentration of 1.5 mg/mL with PBS. In each runs 3.2 mg ofIgG was loaded (in 2.16 mL) on the column. After sample loading (˜2 CV),the column was washed with 7.5 CV of PBS to remove excess of protein.Then the column was eluted with 3 CV of elution buffer (0.1 M Citrate,pH=3.5) to recover the target IgG. The gel was regenerated with 7.5 CVPBS before the next affinity purification cycle. 2.5 mL fractions werecollected and analyzed for IgG content by Abs 280.

When DAP is reacted with 3.2 mg of IgG in solution prior to contactingwith the Biotin-Agarose we recovered 1.2 mg of IgG compared to the 0.35mg IgG which was recovered on average in four repeated cycles withimmobilized DAP.

The above results thus illustrate the advantageous effect of using a DAPaccording to the invention compared to conventional chromatography.

Example 8 Recovery of IgG Using Dual Affinity Chromatography

The purified DAP molecules from examples 5a and 5b were evaluated in ageneric purification assay.

The experiments were conducted at room temperature using an Aktaexplorer system. 0.6 mL solid phase material was packed at a flow rateof 1.2 mL/min in an empty glass column (6.6×100 mm) equipped with anadjustable flow adaptor (Omnifit). The column was packed in 0.1 M sodiumphosphate, 0.15 M NaCl, pH=7.2 (PBS) and allowed to equilibrate with 10column volumes PBS followed by 3 column volumes 0.1 M citrate, pH=3.5and finally 10 column volumes PBS before use.

ZZ-CBD-CDB and CBD-ZZ-CBD were analyzed using a column packed withcellulose. 1.2 g Avicel (Merck product no. 1.02331) was suspended in 8ml PBS in a test tube and the suspension was allowed to settle for 30min. Subsequently the fine particles were decanted before the column waspacked.

AMG-Z and AMG-ZZ were evaluated using a column packed withAcarbose-agarose. Approx. 0.6 mL of the Acarbose-agarose from Example 9was transferred to the column and allowed to settle for 10 min beforethe column was packed.

VhhRR6(R2)-Z was analysed using a column packed with RR6-agarose.Approx. 0.6 mL of the Reactive Red-agarose from Example 9 wastransferred to the column and allowed to settle for 10 min before thecolumn was packed.

Avidin-Protein A, Avidin-Affibody and ZZ-Streptavidin were evaluatedusing a column packed with Biotin-agarose. Approx. 0.6 mL ofBiotin-agarose (Sigma B6885) was transferred to the column and allowedto settle for 10 min before the column was packed.

The packed column was operated at a flow rate of 0.6 mL/min. Buffer Awas 0.1 M sodium phosphate pH 7.2, 0.15 M NaCl and buffer B was 0.1 MCitric acid pH 3.5. The column was initially washed with 10 columnvolumes buffer A before 0.6 mL sample was injected. The column waswashed with 7.5 column volumes buffer A and the bound target protein waseluted with 5 column volumes buffer B. The column was finallyregenerated with 10 column volumes buffer A. Detection was at 280 nm.The data were evaluated by determining the height of the peak observedduring elution.

Purified DAP (8 nmole) was mixed with IgG (code X0903, DAKO NS, 8 nmole)and water was added ad 660 μL. The reaction mixture was incubated on amagnetic stirrer for 10 minutes before it was injected onto the column.The solution of target protein was prepared as a 2 mg/mL IgG solution inwater. The following sequence of injections was carried out in allexperiments: Water; target protein (7.1 nmole); target protein and DAPreaction mixture; and finally 10 times subsequent injections of targetprotein (7.1 nmole).

The column employed for evaluating the Protein A—Avidin DAP molecule wassubsequently used for assessing the effect of changing the load oftarget protein by varying the injection volume. Four injections weremade: (0.6; 0.45; 0.3; 0.15) mL of the same IgG solution (12 μM). Theresults showed that the height of the peak observed during elution wasalmost constant whereas the height of peak observed in the flow throughdecreased markedly as the column load was lowered (Table 5). Theseresults are in accordance with the nature of affinity chromatography anddemonstrate that the applied approach of evaluating the data by usingthe height of the peak observed during elution is valid.

TABLE 5 Peak heights determined from injections of different volumes ofIgG Injection volume Peak height of flow through Peak height eluate mLmAU mAU 0.6 154 126 0.45 116 124 0.3 61 120 0.15 14 109

The non-specific binding of target protein to the column was evaluatedby injecting water and subsequently the target protein before the DAPmolecule was introduced to the column material. The peak heightsobserved from injections of water and IgG were comparable in all theexperiments performed. This demonstrates that the peak observed duringelution is unaffected of potential non-specific binding of targetprotein to the column. Thus the peak observed during elution is ameasure of the recovered amount of target protein from thenon-covalently immobilized DAP.

The ability of the DAP molecules to recover the target protein wasanalyzed by comparing the chromatograms obtained from injection ofwater, target protein and the target protein/DAP reaction mixture. Theresults are shown in Table 6. Only two DAP molecules were unable torecover the target protein. 1) VhhRR6(R2)-Z did not recover IgG, whichmay be explained by results from the Biacore analysis showing that thebinding between DAP and ligand is broken completely as soon as theinjection is stopped (Table 1 footnote). This indicates that the DAPmolecule is quickly released from the solid phase and thus not suitablefor affinity chromatography. 2) The ZZ-streptavidin DAP did not recoverIgG which is likely explained by a blocking of the biotin binding sitesdue to the reaction with endogenous biotin present in the fermentationbroth. Thus this ZZ-streptavidin preparation is likely not to bind tothe solid phase.

TABLE 6 Ability of DAP molecules to recover the target protein Bindingand elution of DAP Column materiale target protein AMG-ZZAcarbose-agarose + AMG-Z Acarbose-agarose + ZZ-CBD-CBD Cellulose +CBD-ZZ-CBD Cellulose + VhhRR6(R2)-Z Reactive red-agarose −ZZ-streptavidin Biotin-agarose − Affibody(IgG)-avidin Biotin-agarose +ProteinA-avidin Biotin-agarose +

The leakage of DAP from the column was assessed by 10 consecutiveinjections of the target protein following the initial injection of theDAP/target protein reaction mixture. The heights of the peaks observedduring elution were determined and the relative responses werecalculated relative to the first injection of IgG. The relativeresponses were plotted as a function of the injection number and therelative decrease in peak height was calculated by linear regression.The results from the different DAP—ligand combinations are shown inTable 7 together with the dissociation constants.

TABLE 7 Dissociation constants and relative leakage Rel dec- rease DAPK_(D,t) M K_(D,s) M K_(D,t)/K_(D,s), %/inj r² AMG-ZZ 1 × 10⁻⁹ 4 × 10⁻⁷ 3× 10⁻³ −1.1 0.8 AMG-Z 4 × 10⁻⁹ 2 × 10⁻⁷ 2 × 10⁻² −1.7 1.0 ZZ-CBD-CBD^(a) 7 × 10⁻¹¹ 10⁻⁶   7 × 10⁻⁵ −1.7 1.0 CBD-ZZ-CBD^(a) 1 × 10⁻⁹ 10⁻⁶   1 ×10⁻³ −1.7 0.9 Affibody(IgG)-avidin^(b)  7 × 10⁻¹³ 10⁻¹⁵* 7 × 10²  −0.2 0.3^(c) Protein A-avidin^(b)  2 × 10⁻¹¹ 10⁻¹⁵* 2 × 10⁴  −0.7 0.9^(a)The dissociation constant (K_(D)) of CBDs binding to cellulose isgenerally considered to be ≈10⁻⁶M (Linder et al, Biotechnology andBioengineering, Vol. 60, No. 5, Dec. 5, 1998). ^(b)The dissociationconstant (K_(D)) of avidin binding to biotin is well known to be 10⁻¹⁵ M(Green, N. (1963). Biochem J, 89, 585-591). ^(c)The correlationcoefficient reflects that the calculated leakage is mainly determined bya single point. The relative leakage is −0.05% with r² = 0.0 if thispoint is removed.

The results in Table 7 show purification schemes of the same targetmolecule (IgG) using various compositions of DAP molecules. It isconcluded that the most efficient DAP molecules in affinitychromatography are those with tighter binding to the ligand on thematrix, i.e. those having a relative K_(D,t)/K_(D,s)>10⁰=1.

Specifically, the strong binding towards the column provided by theavidin-biotin bond prevents leakage of the bound DAP molecules.

Example 9 Preparation of Functionalized Resins Materials

-   Resin: Mini-Leak-Low (loading 2-5 mM, Kem-En-Tec).-   Ligands: 1,4-Diaminobutane ([110-60-1], Sigma-Aldrich, D13208),    Reactive Red 6 (Cherry red #14, Grateful Dyes inc.), Acarbose    ([56180-94-0] Sigma-Aldrich, A8980).-   Coupling buffer: 0.5M K₂HPO₄—pH 8.5-   Washing buffer: 0.5M K₂HPO₄—pH 7.0-   Blocking buffer 0.1M Ethanolamine in Milli-Q water

Preparation of an RR6-Agarose Resin

Resin (10 ml, suspended) was washed 2× with destilled water and thewater removed by filtration. 1,4-Diaminobutane (2.0 mL) was dissolved incoupling buffer (20 mL) and the resin was added slowly with gentleshaking. The resin was left shaking overnight at RT, whereupon it waswashed with coupling buffer and sucked dry.

Reactive Red 6 (15.9 g) was dissolved in coupling buffer (50 mL) and tothis solution, the amino-functionalized resin was added slowly withshaking. Again the resin was left shaking overnight at RT. After washingwith water and washing buffer, the resin was transferred to blockingbuffer (20 mL) and shaken for 2 h. Finally the resin was washed in wateruntil the filtrate was colorless, and the red resin was suspended in 30%ethanol in Milli-Q water.

Preparation of an Acarbose-Agarose Resin

Resin (10 ml, suspended) was washed 2× with destilled water and thewater removed by filtration. Acarbose (500 mg) was dissolved in couplingbuffer (20 mL) and the resin was added slowly with gentle shaking. Theresin was left shaking overnight at RT, whereupon it was washed withwater and washing buffer.

The resin was transferred to blocking buffer (20 mL) and shaken for 2 h.Finally the resin was washed in water, and the resulting resin wassuspended in 30% ethanol in Milli-Q water.

1-18. (canceled)
 19. A process for purification of a target biomolecule,comprising the steps: (a) contacting (i) a target biomolecule, (ii) adual affinity polypeptide, and (iii) a solid support comprising acatching ligand or dual affinity polypeptide binding site, wherein theratio between the equilibrium dissociation constants of the dualaffinity polypeptide, [K_(D,t)/K_(D,s)], is at least 10⁰ at standardconditions; and (b) recovering the target biomolecule by elution,wherein the target affinity polypeptide and the dual affinitypolypeptide are contacted in solution before the mixture is contactingthe solid support comprising a catching ligand or dual affinitypolypeptide.
 20. The process according to claim 19, wherein the solidsupport is selected from the group comprising solid phase matrices andparticles.
 21. The process according to claim 19, wherein the dualaffinity polypeptide has an equilibrium dissociation constant, K_(D,t)towards the target biomolecule in the range from 10⁻² to 10⁻¹³ M, moreparticularly from 10⁻⁴ to 10⁻¹³ M, preferably in the range from 10⁻⁶ to10⁻¹³ M and an equilibrium dissociation constant, K_(D,s) towards thecatching ligand in the range from 10⁻⁹ to 10⁻¹⁶ M, preferably in therange from 10⁻¹¹ to 10⁻¹⁶ M.
 22. The process according to claim 19,wherein the ratio between the equilibrium dissociation constants of thedual affinity polypeptide, [K_(D,t)/K_(D,s)], is at least 10¹, moreparticularly at least 10², more particularly 10³, and even moreparticularly at least 10⁴.
 23. The process according to claim 19,wherein elution of the target is accomplished by changing either of pH,ionic strength, or content of chaotropic ions in solution, or anycombinations thereof.
 24. The process according to claim 19, wherein thedual affinity polypeptide is a fusion polypeptide, preferably selectedfrom the group consisting of protein A, antibodies, antibody fragments,protein A fragments, protein A derived IgG binding domains, lipocalins,lectins.
 25. The process according to claim 19, wherein the ligandbinding part of the dual affinity polypeptide is selected from the groupconsisting of avidin, streptavidin, neutravidin, steroid receptor,antibody, antibody fragment, lipocalins, lectins, amyloglucosidase,cellulose binding domains.
 26. The process according to claim 24,wherein the antibody is selected from the group consisting of llama andcamel antibodies.
 27. The process according to claim 24, wherein thefusion polypeptide is made by fusion of at least one IgG binding domainof protein A or protein A derived IgG binding domain and at least onebiotin binding domain of avidin, streptavidin, or neutravidin.
 28. Theprocess according to claim 19, wherein the ligand is selected from thegroup consisting of biotin, acarbose, steroids, hapten,epitope-peptides, dyes, and enzyme inhibitors.
 29. The process accordingto claim 27, wherein the catching ligand attached to the solid supportis biotin and the target biomolecule is IgG.
 30. The process accordingto claim 19, wherein the solid support is a solid phase matrix,preferably selected from the group consisting of agar-agar, agaroses,celluloses, cellulose ethers, carboxymethyl cellulose, polyamides,polyvinylalcohols, silicas, and controlled pore glasses.
 31. The processaccording to claim 24, wherein the fusion polypeptide is produced as arecombinant polypeptide in a recombinant host cell.
 32. The processaccording to claim 31, wherein the fusion polypeptide and the targetbiomolecule is expressed in the same type of host cell.
 33. The processaccording to claim 31, wherein the host cell is selected from the groupconsisting bacterial cells, fungal cells, mammalian cells, plant cells,and insect cells.
 34. The process according to claim 19, wherein thedual affinity polypeptide is chemically fused.
 35. A process forpurification of a target biomolecule, comprising the steps: (a)contacting (i) a target polypeptide, (ii) a dual affinity polypeptide,and (iii) a solid support comprising a catching ligand, wherein the dualaffinity polypeptide has an equilibrium dissociation constant, K_(D,t)towards the target biomolecule in the range from 10⁻² to 10⁻¹³ M,preferably from 10⁻⁴ to 10⁻¹³ M, more preferred from 10⁻⁶ to 10⁻¹³ M atstandard conditions, and wherein binding of the dual affinitypolypeptide to the catching ligand on the solid support is provided bycleavage of a para-substituted benzyl guanine resulting in a thioetherbond; and (b) recovering the target biomolecule by elution, where thetarget polypeptide and the dual affinity polypeptide are contacted insolution before the mixture is contacting the solid support comprising acatching ligand.